CN103403557B - Microfluidic processing of target species in ferrofluids - Google Patents
Microfluidic processing of target species in ferrofluids Download PDFInfo
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- CN103403557B CN103403557B CN201180063125.3A CN201180063125A CN103403557B CN 103403557 B CN103403557 B CN 103403557B CN 201180063125 A CN201180063125 A CN 201180063125A CN 103403557 B CN103403557 B CN 103403557B
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- B03C1/23—Magnetic separation acting directly on the substance being separated with material carried by oscillating fields; with material carried by travelling fields, e.g. generated by stationary magnetic coils; Eddy-current separators, e.g. sliding ramp
- B03C1/24—Magnetic separation acting directly on the substance being separated with material carried by oscillating fields; with material carried by travelling fields, e.g. generated by stationary magnetic coils; Eddy-current separators, e.g. sliding ramp with material carried by travelling fields
- B03C1/253—Magnetic separation acting directly on the substance being separated with material carried by oscillating fields; with material carried by travelling fields, e.g. generated by stationary magnetic coils; Eddy-current separators, e.g. sliding ramp with material carried by travelling fields obtained by a linear motor
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- B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
- B03C1/00—Magnetic separation
- B03C1/02—Magnetic separation acting directly on the substance being separated
- B03C1/28—Magnetic plugs and dipsticks
- B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
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- B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
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- B03C1/32—Magnetic separation acting on the medium containing the substance being separated, e.g. magneto-gravimetric-, magnetohydrostatic-, or magnetohydrodynamic separation
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44786—Apparatus specially adapted therefor of the magneto-electrophoresis type
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- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
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Abstract
Disclosed are systems, devices, methods, and other implementations, including a device to detect at least one target species in a sample, with the device including a microfluidic channel configured to receive the sample containing the at least one target species and a biocompatible ferrofluid in which the at least one target species is suspended, a detector to determine the at least one target species in the sample, and at least two of electrodes positioned proximate the microfluidic channel, the at least two electrodes configured to generate controllable magnetic forces in the sample containing the ferrofluid when a controllable at least one electrical current is applied to the at least two electrodes. The generated controllable magnetic forces cause the at least one target species to be directed towards the detector. Also disclosed are devices for separating target species in a ferrofluid, and for focusing target species suspended in a ferrofluid.
Description
The cross reference of related application
This application claims on Dec 7th, 2010 submit to, PCT application PCT/US10/59270, the U.S.Provisional Serial 61/267 that exercise question was submitted to for " n cell undertaken by biocompatible ferrofluid is operated and sorting " and on Dec 7th, 2009, the U.S.Provisional Serial 61/407 that on October 28th, 163 and 2010 submits to, the right of priority of 738, its all the elements this by reference entirety be incorporated to.
Field
The disclosure relates generally to the miniflow process of ferrofluid, comprises the ferrofluid comprising plurality of target material (such as biological target substance).More particularly, the disclosure relates to device, system and method, to carry out these operations, as being separated plurality of target material in biocompatible ferrofluid, in biocompatible ferrofluid, concentrating target substance, detecting target substance etc. in the sample to which.
Background
The early diagnosis relating to the disease (as metastatic carcinoma or low-level bacteremia) of rare cell in blood needs with the accurate measurements of some genetic disease state (as sickle cell anemia) to be separated fast and accurately, sorting and guide target cell type be towards sensor surface.In this respect, cell manipulation, be separated and be sorted in cancer diagnosis (Dittrich etc., 2006,
nat Rev Drug Discovery5:210-218), pathogen detection (Beyor etc., 2008,
blamed Microdevices10:909-917) and genomic testing (Kamei etc. 2005,
biotned Microdevices7:147-152; Cheong etc., 2008,
lab Chipapplication potential is found just more and more in various biologicall tests 8:810-813) under background.
There is various noncontact microoperation method, comprise light tweezer (Ashkin etc., 1987,
nature330:769-771; Chian et al., 2005,
nature436:370-372), dielectrophoresis (DEP) (Hughes, 2002,
electrophoresis23:2569-2582), based on magnetic bead separation vessel (Lee et al., 2001,
appl Phys Lett79:3308-3310; Yan et al., 2004,
phys Rev E70:011905) and determinacy fluid dynamics (Davis et al., 2006,
proc Natl Acad Sci USA103:14779-14784).But a lot of existing method can not reliably realize fast, high flux and resolution, simultaneously low cost (Dufresne et al., 1998,
rev Sci Instrum69:1974-1977; Kremser et al., 2004,
electrophoresis25:2282-2291; Cabrera et al., 2001,
electrophoresis22:355-362).Light tweezer provides operation single celled high resolving power and sensitivity, although these operations may cause sample heat (Liu et al., 1995,
biophys J68:2137-2144), and be usually limited to minimum region (Ashkin et al., 1987,
science235:1517-1520).Holographic scheme light tweezer scope has been extended to recently touch simultaneously tens cells (Applegate et al., 2004,
optical Express12:4390-4398), although total flux is still very low.
Such as, based on the scheme of electric field, DEP, provide and realize comprehensive, the effective device of cost for operating the potential of various kinds of cell simultaneously.But, their performance sensitive ground depend on the electrical property of concrete liquid medium, particle shape and its effective dielectric constant (Pethig et al., 1997,
trends Biotechnol15:426-432).DEP device operation scheme and work ionic medium need careful optimization for each different cell type, with reach minimizing heating (Menachery et al., 2005,
nanoBiotechnology152:145149; Muller, et al., 2003,
iEEE Eng Biol Med Mag22:51-61) and by polarization minimize (Sebastian et al., 2006,
j Micromech Microengspendable balance between needs 16:1769-1777).Non-electric field overcomes these challenges by using magnetic field for the Beads enrichment target molecule of using function and cell.But the unfavorable aspect of this technology is long incubation time and clean cycle, and the difficulty of removal posteriority mark (Gijs 2004,
microfluidics Nanofluidics1:22-40).By Davis etc. (Davis et al., 2006,
proc Natl Acad Sci USAdeterminacy hydrodynamic method 103:14779-14784) shown can realize high-resolution separation, and does not use any electromagnetic field.But the high flux of this device needs high-resolution photoetching over a large area, each installation cost is made to keep high.
In biological medicine, the most common application of ferrofluid relates to the soliquid of the high dilution of magnetic nanoparticle.Its most widely commercial use be MRI contrast agent (Kim et al., 2005,
j Magn Magn Mater289:328-330).When suitably using target antibody dressing, they can also be used for cancer hyperthermia therapy or as sensor detect pathogen (Scherer et al., 2005,
brazilian J Phys45:718-727).
Although these development during ferrofluid uses provide the chance much in medicine and diagnosis, this area still needs biocompatible ferrofluid to be used for the controlled operation of particulate and living cells and the miniflow platform of quick separating.
General introduction
Describe the device being separated in the particulate samples suspended in biocompatible ferrofluid.In some embodiments, device comprises microchannel, and it has sample inlet, at least one outlet and the length between sample inlet and at least one outlet, and wherein sample can be added to sample inlet, and flows at least one outlet along length.Device comprises multiple electrode, and wherein microchannel length crosses multiple electrode, and comprises the power supply applying electric current to multiple electrode further, produces magnetic field model with the length along microchannel.In some embodiments, interelectrode interval increases gradually.In some embodiments, interelectrode interval is reduced gradually.In some embodiments, multiple electrode comprises at least one electrode layer.In some embodiments, multiple electrode comprises multiple electrode layer.In some embodiments, multiple electrode layer is right angle mode substantially.In some embodiments, multiple electrode comprises concentric circles pattern.In some embodiments, the wall of microchannel length comprises region that is small-sized, carinate, reeded, fluted or that tilt.In some embodiments, microchannel length with about 0-360(and more particularly, 0-90 degree) between angle cross multiple electrode at least partially.In some embodiments, particle is living cells.
Also describe the system being separated at least one target from the sample be suspended in biocompatible ferrofluid.System comprises microchannel, and it has sample inlet, at least one outlet and the length between sample inlet and at least one outlet, and wherein sample can be added to sample inlet, and flows at least one outlet along length.System also comprises multiple electrode, and wherein microchannel length crosses multiple electrode, and when electric current is applied to electrode, produces the magnetic field model along microchannel length further.System comprises at least one target in the sample be suspended in biocompatible ferrofluid further, wherein when at least one target along microchannel length at least partially by time, at least one target is separated from remaining sample.In one embodiment, biocompatible ferrofluid comprises appropriate ionic species to control osmotic pressure on cell to promote cell continuation.In some embodiments, biocompatible ferrofluid comprises the citric acid salt concentration of about 5-200mM.In some embodiments, biocompatible ferrofluid comprises the citric acid salt concentration of about 40mM.In some embodiments, biocompatible ferrofluid has the pH of about 7.4.In some embodiments, at least one target based target size separation.In some embodiments, at least one target based target shape is separated.In some embodiments, at least one target based target resilient separation.In some embodiments, target is separated by being directed to selected outlet.In some embodiments, target is captured based on electrode gap.In some embodiments, at least one target is cell.In some embodiments, at least one target is particle.
Also describe the method for separating at least one cell type.Method comprises the following steps, two or more cell types are suspended in biocompatible ferrofluid to form sample, sample is made to pass through to cross the microchannel of multiple electrode, apply electric current to produce magnetic field model along microchannel length to multiple electrode, and enter at least one exit passageway based on the difference sorting cells of cell size, shape and flexible at least one.In one embodiment, cell separation efficiency is at least 90%.In some embodiments, the size resolution of separation is less than about 10 μm.In some embodiments, cell be less than in about 1 minute be separated.
In some embodiments, system described herein, device, method and product relate to miniflow platform, and biocompatible ferrofluid is used for controlled operation and the quick separating of particulate and living cells by it.In some embodiments, system as herein described, device, method, and product can by biocompatible concentrated ferrofluid carry out cell high flux operation, unmarked classified and separated.The biocompatibility of ferrofluid based on ionic surface active agent, the effective balance of such as citrate, or concentration.Biocompatibility needs neutral pH usually, osmotic pressure enough on cell, and stable ferrofluid (such as, too many ion content thing can make suspending liquid instability).This platform utilizes the difference of particle size, shape, elasticity, form etc., with realize fast be effectively separated.The separation based on size of microballoon is used to show being less than in such as about 45 seconds, the separation efficiency of about 99% and sub-10 μm of resolution.System as herein described, device, method and product also provide being separated of erythrocytic continued operation that to carry out living from sickle cell and bacterium and Shape-based interpolation.Ferromagnetic microfluid system described herein, device, method and product are delivered to sensor array by quick separating with by target cell in raji cell assay Raji, significantly reduce incubation time and increase diagnostic sensitivity.
Therefore, in some embodiments, the system being separated the multiple target substance be suspended in biocompatible ferrofluid is disclosed.System comprises microchannel, it comprises at least one sample inlet and at least one outlet, microchannel has the length extended between at least one sample inlet and at least one outlet, microchannel is configured to accept continuous print sample stream substantially from least one sample inlet, passage is configured to make sample flow at least one outlet along passage length, and sample comprises plurality of target material and biocompatible ferrofluid.System also comprises multiple electrode, wherein at least partially nearest through multiple electrode of microchannel length, and power configuration is controllably apply at least one electric current to multiple electrode, with the controlled generation magnetic field model of passage length at least partially along microchannel, cause at least two kinds of separation in sample in plurality of target material.
The embodiment of system can comprise any feature described in the disclosure, comprises any following features.
Power supply can be configured to the extra at least one electric current that controllably applies to produce magnetic force component and magnetic moment component.
The plurality of target material be separated can be separated with on the direction of the direction perpendicular of continuous print sample stream substantially in microchannel.
Substantially continuous print sample stream can pass through such as, forcing pump, syringe pump, peristaltic pump, vacuum plant, gravity, and/or at least one of capillary force provides.
Power supply can be configured to basis such as, and at least one of selected amplitude, selected frequency and/or selected phase place applies electric current.The separation of at least two kinds of target substances of sample can at least partly based on one or more of the amplitude such as selected by electric current, selected frequency and/or selected phase place.
Multiple interelectrode interval can increase gradually along its length.
Interval between at least some of multiple electrode can be reduced gradually along its length.
Multiple electrode can be arranged in electrode layer.
Multiple electrode can be arranged in multiple electrode layer.
Multiple electrode layer can the mode arrangement of perpendicular.
Multiple electrode layer can concentrically ringed mode arrangement.
The wall of microchannel can limit such as small-sized, carinate, reeded, the fluted or region that tilts.
The length of microchannel can pass at least partially nearest of multiple electrode with the angle between about 0-90 degree.
Plurality of target material can comprise the material of at least one based on cell.Material based on cell can comprise following one or more, such as, and leucocyte, red blood cell, tumour cell and/or the cell based on bacterium.
In some embodiments, the device being separated the multiple target substance be suspended in biocompatible ferrofluid is disclosed.Device comprises microchannel, comprise at least one sample inlet and at least one outlet, microchannel has the passage length extended between at least one sample inlet and at least one outlet, microchannel is configured to accept continuous print sample stream substantially from least one sample inlet, and be further configured to make sample along passage length flow at least one outlet.Sample comprises plurality of target material and biocompatible ferrofluid.Device also comprises multiple electrodes of the contiguous microchannel in position, and multiple electrode is configured to when electric current is applied to multiple electrode, produces the magnetic field model at least partially of the passage length along microchannel.Magnetic field model is configured to, when sample stream is advanced along at least part of microchannel, cause at least two kinds of separation of plurality of target material in sample stream.
The embodiment of device can comprise any feature described in the disclosure, comprises any above-mentioned characteristic sum hereinafter described feature relevant with system.
Device may further include power supply, is configured to controllably apply electric current controllably to produce magnetic field model to multiple electrode.
Multiple electrode can be configured to produce controlled magnetic force component and controlled magnetic moment component.
Multiple electrode can be configured to cause at least two kinds of target substances being separated with on the direction of the direction perpendicular of continuous print sample stream substantially in microchannel.
Device may further include the stream generation unit being configured to produce continuous stream substantially.Stream generation unit can comprise following at least one, such as, forcing pump, syringe pump, peristaltic pump, vacuum plant, is made it the structure that flows by gravity, and/or produces the device of capillary force.
Biocompatible ferrofluid can comprise appropriate ionic species to control osmotic pressure on cell to promote the continuation of cell.Biocompatible ferrofluid can comprise the citric acid salt concentration of about 5-200 mM.Biocompatible ferrofluid can comprise the citric acid salt concentration of about 40 mM.Biocompatible ferrofluid can comprise the ionic strength of the about 150mM of design, thus biocompatible ferrofluid is isotonic and is applicable to maintaining the eukaryotic of living.
Biocompatible ferrofluid can have the pH of about 7.4.
Described at least two kinds of materials can based target size be separated.
Described at least two kinds of materials can based target shape be separated.
Described at least two kinds of materials can based target resilient separation.
Described at least two kinds of materials can based target form fractionation.
Described at least two kinds of materials can be captured based on following one or more, such as, electrode separation, apply electric current frequency and/or apply the phase place of electric current.
In some embodiments, the method being separated plurality of target material is disclosed.The entrance that method is included in microchannel accepts to comprise the sample of the continuous print substantially stream of the plurality of target material be suspended in biocompatible ferrofluid, this sample is passed through along microchannel, and applies the controlled electric current of at least one to the electrode of multiple positions adjacent channel.Current arrangements is controllably produce magnetic field model along the passage length at least partially of microchannel, with cause at least two kinds of plurality of target material in sample separated.
The embodiment of method can comprise any feature described in the disclosure, comprises any above-mentioned characteristic sum hereinafter described feature relevant with system and device.
Method at least two kinds of may further include in the plurality of target material that sorting is separated enter at least one output channel, and it is based on the difference of following at least one, such as, and cell size, shape, elasticity, and/or form.
Apply the controlled electric current of at least one can comprise and controllably apply at least one electric current to produce magnetic force component and magnetic moment component.
Controllably applying electric current to multiple electrode causes at least two kinds of plurality of target material being separated with on the direction of the direction perpendicular of continuous print sample stream substantially in microchannel.
Accept continuous print sample stream substantially and comprise the following at least one of use from external source generation pressure to provide continuous print sample stream, such as, forcing pump, syringe pump, peristaltic pump, vacuum plant, the structure of the pressure that gravity is assisted can be produced, and/or produce the device of capillary force.
Controllably applying at least one electric current can comprise according to one or more following applying electric currents, such as, selected amplitude, institute's selected frequency and/or selected phase place, wherein the separation of at least two kinds of target substances of sample is at least partly based on one or more of such as amplitude, institute's selected frequency and/or selected phase place selected by electric current.
In some embodiments, the device concentrating at least one target substance be suspended in biocompatible ferrofluid is disclosed.Device comprises microchannel, is configured to the sample accepting to comprise at least one target substance and biocompatible ferrofluid, and at least one target substance in the sample of acceptance is concentrated in the inlet flow with relevant input width substantially.Equipment also comprises at least two electrodes, and the contiguous microchannel in position, described at least two electrodes are configured to, when controlled electric current puts at least two electrodes, in the sample comprising ferrofluid, produce controlled magnetic force.The controlled magnetic force produced causes at least one target substance to focus in produced stream, and its width is narrower than the relevant input width of inlet flow.
The embodiment of device can comprise any feature described in the disclosure, comprises any above-mentioned characteristic sum hereinafter described feature relevant with system, first device and method.
At least two electrodes of the contiguous microchannel in described position can be configured to conduct the electric current of controlled supply and produce controlled magnetic force, and this is at least partly according to physical characteristics of at least two electrodes.Described at least two electrodes comprise the structure with following at least one, such as, the substantially parallel layout of the one or more substantially straight shape of at least two electrodes, one or more corrugated shapes substantially of at least two electrodes, at least two electrodes, and/or the location of at least two electrodes taper substantially, wherein said at least two electrodes are close to each other gradually.
Described at least two electrodes can be configured to cause the space in the microchannel of at least one target substance inflow between top and at least two electrodes.
At least two electrodes can be configured at least two magnetic waves producing backward-travelling wave field (reverse traveling field), are concentrated to border immediate vicinity between at least two magnetic waves produced to cause at least one target substance.
At least two electrodes can comprise the electrode of multiple substantially parallel layout.
Described at least two electrodes can comprise the array of electrode, the electrode tapered oriented arrangement substantially that at least some first electrode of array is relatively adjacent, be configured to move closer to adjacent electrode to make the first electrode, electrod-array be configured to produce magnetic force, with cause produce at least one target substance stream up and adjacent electrode between formed.
At least two electrodes can be configured to, when described in the controllable current with dependent phase is applied to during at least two electrodes, produce controlled magnetic force in the sample to which.The dependent phase of at least one electric current can be different from the dependent phase of another electric current.
At least one target substance comprises at least two kinds of target substances.Multiple electrode can be further configured to cause at least two kinds of plurality of target material in the produced stream concentrated separated.
Interval between at least two electrodes can increase gradually.
Interval between at least two electrodes can be reduced gradually.
At least two electrodes can be arranged at least one electrode layer.
At least two electrodes can be arranged in multiple electrode layer.
The length of microchannel can with the angle between about 0-90 degree through at least two contiguous electrodes at least partially.
Described at least one target substance comprises the target substance based on cell.
In some embodiments, the system concentrating at least one target substance be suspended in biocompatible ferrofluid is disclosed.Described system comprises microchannel, is configured to the sample accepting to comprise at least one target substance and biocompatible ferrofluid, and at least one target substance in the sample of acceptance is concentrated in the inlet flow with relevant input width substantially.System also comprises at least two electrodes, and the contiguous microchannel in position, described at least two electrodes are configured to, when controlled electric current puts at least two electrodes, in the sample comprising ferrofluid, produce controlled magnetic force.The controlled magnetic force produced causes at least one target substance to focus in produced stream, and its width is narrower than the relevant input width of inlet flow.System comprises power supply further controllably to apply controlled electric current at least two electrodes.
The embodiment of system can comprise any feature described in the disclosure, comprise any with the first system, the relevant above-mentioned characteristic sum hereinafter described feature of apparatus and method.
Power supply can be configured to apply electric current, and it comprises amplitude selected separately, selected relevant respective frequency, and/or selected relevant respective phase place.Concentrating of at least one target substance can at least partly based on such as, institute apply electric current respective selected by amplitude, selected frequency and/or selected phase place.
System comprises pressure generating unit further, to provide inlet flow, pressure generating unit comprises following at least one, such as, forcing pump, syringe pump, peristaltic pump, vacuum plant, the structure of the pressure that gravity is assisted can be produced, and/or produce the device of capillary force.
Space in the microchannel that controlled magnetic force can cause the stream of the generation of at least one target substance to be pushed between top and at least two electrodes.
At least two electrodes of the contiguous microchannel in position can be configured at least two magnetic waves producing backward-travelling wave field, cause at least one target substance to be concentrated to border immediate vicinity between at least two magnetic waves produced.
At least two electrodes being configured at least two magnetic waves producing relevant backward-travelling wave field can comprise the electrode of multiple substantially parallel layout.
Described at least two electrodes can comprise the array of electrode, comprise multiple first electrodes of relatively adjacent electrode tapered oriented arrangement substantially, adjacent electrode is moved closer to make the first electrode, electrod-array be configured to produce magnetic force, with cause produce at least one target substance stream up and adjacent electrode between formed.
At least two kinds of electrodes can be configured to, when the controlled electric current with dependent phase is applied at least two electrodes, in the sample comprising ferrofluid, produce controlled magnetic force.The dependent phase of at least one electric current can be different from the dependent phase of another electric current.
In some embodiments, the method concentrating at least one target substance in microchannel is disclosed.Described method comprises the sample accepting to comprise at least one target substance be suspended in biocompatible ferrofluid, and at least one target substance in the sample of acceptance is concentrated in the inlet flow with relevant input width substantially.At least two electrodes that method also comprises the contiguous microchannel to position controllably apply at least one electric current, to produce controlled magnetic force in the sample comprising ferrofluid passage.Magnetic force causes at least one target substance to focus in produced stream, and its width is narrower than the relevant input width of inlet flow.
The embodiment of method can comprise any feature described in the disclosure, comprise any with system, the device above-mentioned characteristic sum hereinafter described feature relevant with first method.
Controllably apply at least one electric current and can comprise following one or more controllably selecting at least one electric current, such as, relevant respective amplitude, relevant respective frequency and relevant respective phase place.At least one target substance concentrate can at least partly based on, such as, the separately selected amplitude of at least one electric current applied, frequency selected separately and/or separately selected by phase place.
Method may further include and uses pressure generating unit to provide inlet flow, pressure generating unit comprises following at least one, such as, forcing pump, syringe pump, peristaltic pump, vacuum suction device, the structure of the pressure that gravity is assisted can be produced, and/or produce the device of capillary force.
At least two electrodes are configured to conduct the electric current that controllably applies to produce controlled magnetic force, the space in the microchannel causing the stream of at least one target substance produced to be pushed between top and at least two electrodes.
In some embodiments, the device detecting at least one target substance in sample is disclosed.Device comprises microchannel, its be configured to accept to comprise at least one target substance and wherein at least one target substance be suspended in the sample of biocompatible ferrofluid wherein, the detecting device of at least one target substance in working sample, and at least two electrodes of the contiguous microchannel in position.At least two electrodes are configured to, when at least one controlled current flow is applied at least two electrodes, in the sample comprising ferrofluid, produce controlled magnetic force, and the controlled magnetic force produced causes at least one target substance guiding detecting device.
The embodiment of device can comprise any feature described in the disclosure, comprises any above-mentioned characteristic sum hereinafter described feature relevant with system, apparatus and method.
At least two electrodes can comprise electrod-array, and at least some electrode is arranged taper location substantially with relative adjacent electrode, thus at least some electrode moves closer to their adjacent electrode.
At least two electrodes can be configured to, when the controlled electric current comprising dependent phase is applied at least two electrodes, in the sample comprising ferrofluid, produce controlled magnetic force.The dependent phase of at least one electric current can be different from the dependent phase of another electric current.
Sample can comprise plurality of target material, and device may further include electrode group, be configured to when controlled electric current is applied to this electrode group, produce controlled magnetic field model at least partially along microchannel length, cause at least two kinds of plurality of target material in sample separated.
Microchannel can be configured to accept the sample stream from external sample source.
Detecting device can comprise the electrode at a pair interval of the electric capacity measured in microchannel, and the qualification unit of existence based on measured electric capacity determination at least one target substance.
Be configured to determine qualification unit that at least one target substance exists can be configured to cause based on the existence due at least one target substance measured by microchannel in the change of electric capacity determine the existence of at least one target substance.
Detecting device may further include capture region, comprise and be configured to material interactional with one of plurality of target material, capture region is positioned at the microchannel downstream of the electrode pair at interval, and another downstream electrode at interval being arranged in capture region is to measure the electric capacity of microchannel.Qualification unit can be configured to the electric capacity determination at least one target substance measured based on the electrode pair at the interval initial number in the electrode pair at interval, and based at another end number to the electric capacity determination at least one target substance that the electrode place at interval is measured, and determine whether the level of at least one target substance of catching in captured region exceeds predetermined threshold value based on the difference between initial and end number at least partly.
The test set that the order that detecting device can comprise series connection is arranged, the test set that the order of each series connection is arranged comprises the electrode at first pair of interval to measure the electric capacity in microchannel, capture region comprises and is configured to material interactional with one of plurality of target material, capture region is positioned at the downstream of the electrode at first pair of interval, and the electrode at second pair of interval is arranged in the downstream of capture region to measure the electric capacity of microchannel.Device can also comprise qualification unit, with at each test set place based on the initial number of electric capacity determination at least one target substance at the electrode place at first pair of interval that the electrode place at first pair of interval is measured, and based on the end number of electric capacity determination at least one target substance at the electrode place at second pair of interval that the electrode place at second pair of interval is measured, and determine whether the level of at least one target substance of catching in captured region exceeds predetermined threshold value based on the difference between initial and end number at least partly.
Detecting device can comprise the electrode at a pair interval of the impedance of measuring in microchannel, and the qualification unit of existence based on measured impedance determination at least one target substance.
Be configured to determine qualification unit that at least one target substance exists can be configured to cause based on the existence due at least one target substance measured by microchannel in the change of impedance determine the existence of at least one target substance.
Detecting device may further include capture region, comprise and be configured to material interactional with one of plurality of target material, capture region is positioned at the microchannel downstream of the electrode pair at interval, and another downstream electrode at interval being arranged in capture region is to measure the impedance of microchannel.Qualification unit can be configured to the impedance determination at least one target substance measured based on the electrode pair at the interval initial number in the electrode pair at interval, and based on the impedance determination at least one target substance measured the electrode place at interval at another at another end number to the electrode place at interval, and determine whether the level of at least one target substance of catching in captured region exceeds predetermined threshold value based on the difference between initial and end number at least partly.
The test set that the order that detecting device can comprise series connection is arranged, the test set that the order of each series connection is arranged comprises the electrode at first pair of interval to measure the impedance in microchannel, capture region comprises and is configured to material interactional with one of plurality of target material, capture region is positioned at the downstream of the electrode at first pair of interval, and the electrode at second pair of interval is arranged in the downstream of capture region to measure the impedance of microchannel.Device can also comprise qualification unit, with at each test set place based on the initial number of impedance determination at least one target substance at the electrode place at first pair of interval that the electrode place at first pair of interval is measured, and based on the end number of impedance determination at least one target substance at the electrode place at second pair of interval that the electrode place at second pair of interval is measured, and determine whether the level of at least one target substance of catching in captured region exceeds predetermined threshold value based on the difference between initial and end number at least partly.
In some embodiments, the system detecting at least one target substance in sample is disclosed.System comprises microchannel, be configured to accept to comprise the sample that at least one target substance and at least one target substance are suspended in biocompatible ferrofluid wherein, determine the detecting device of at least one target substance in sample, at least two electrodes of the contiguous microchannel in position, at least two electrodes are configured to when controlled at least one electric current puts at least two electrodes, controlled magnetic force is produced in the sample comprising ferrofluid, the controlled magnetic force produced causes at least one target substance to be directed to detecting device, with the power supply controllably applying controlled at least one electric current at least two electrodes.
The embodiment of system can comprise any feature described in the disclosure, comprises any above-mentioned characteristic sum hereinafter described feature relevant with system, apparatus and method.
The power supply controllably applying controlled at least one electric current at least two electrodes can be configured to the electric current applying to have relevant selected separately amplitude, selected frequency and selected phase place, by least one target substance guiding detecting device be wherein at least partly based on applied electric current separately selected by amplitude, selected frequency and selected phase place.
System comprises pressure generating unit further, with sampling stream, pressure generating unit comprise following one or more, such as, forcing pump, syringe pump, peristaltic pump, vacuum suction device, the structure of the pressure that gravity is assisted can be produced, and/or produce the device of capillary force.
In some embodiments, the method detecting at least one target substance in sample is disclosed.Method is included in microchannel the sample accepting to comprise at least one target substance be suspended in biocompatible ferrofluid, to position, at least two electrodes of contiguous microchannel controllably apply at least one electric current, cause at least one target substance to be directed to detecting device to produce controlled magnetic force in the sample comprising ferrofluid passage, and the measurement that the sample in microchannel carries out determined at least one target substance in sample based on detecting device.
The embodiment of method can comprise any feature described in the disclosure, comprises any above-mentioned characteristic sum hereinafter described feature relevant with system, apparatus and method.
Controllably apply at least one electric current and can comprise following one or more controllably selecting at least one electric current, such as, each autocorrelative amplitude, each autocorrelative frequency and/or each autocorrelative phase place.At least partly based on selected separately amplitude, selected frequency and/or the selected phase place of such as applied at least one electric current by least one target substance guiding detecting device.
Method may further include and uses pressure generating unit to be provided in the sample accepted in microchannel, pressure generating unit comprises following at least one, such as, forcing pump, syringe pump, peristaltic pump, vacuum suction device, the structure of the pressure that gravity is assisted can be produced, and/or produce the device of capillary force.
Detecting device can comprise the electrode pair at the interval of the electric capacity measured in microchannel, and determines that at least one target substance can comprise the existence based on measured electric capacity determination at least one target substance.
Detecting device may further include capture region, comprise and be configured to material interactional with one of plurality of target material, capture region is positioned at the microchannel downstream of the electrode pair at interval, and another downstream electrode at interval being arranged in capture region is to measure the electric capacity of microchannel.Determine that the existence of at least one target substance in sample can comprise, the electric capacity determination at least one target substance measured based on the electrode pair at interval is in the initial number of the electrode pair at interval, and based on the electric capacity determination at least one target substance measured the electrode place at interval at another at another end number to the electrode place at interval, and determine whether the level of at least one target substance of catching in captured region exceeds predetermined threshold value based on the difference between initial and end number at least partly.
The test set that the order that detecting device can comprise series connection is arranged, the test set that the order of each series connection is arranged comprises the electrode at first pair of interval to measure the electric capacity in microchannel, capture region comprises and is configured to material interactional with one of plurality of target material, capture region is positioned at the downstream of the electrode at first pair of interval, and the electrode at second pair of interval is arranged in the downstream of capture region to measure the electric capacity of microchannel.Determine the initial number of electric capacity determination at least one target substance at the electrode place at first pair of interval that at least one target substance can be included in each test set place and measures based on the electrode pair at interval, and based on the end number of electric capacity determination at least one target substance at the electrode place at second pair of interval measured the electrode place at interval at another, and determine at each test set place whether the level of at least one target substance of catching in captured region exceeds predetermined threshold value at least partly based on the difference between initial and end number.
Detecting device can comprise the electrode pair at the interval of the impedance of measuring in microchannel, and determines that at least one target substance can comprise the existence based on measured impedance determination at least one target substance.
Detecting device may further include capture region, comprise and be configured to material interactional with one of plurality of target material, capture region is positioned at the microchannel downstream of the electrode pair at interval, and another downstream electrode at interval being arranged in capture region is to measure the impedance of microchannel.Determine that the existence of at least one target substance in sample can comprise, the impedance determination at least one target substance measured based on the electrode pair at interval is in the initial number of the electrode pair at interval, and based on the impedance determination at least one target substance measured the electrode place at interval at another at another end number to the electrode place at interval, and determine whether the level of at least one target substance of catching in captured region exceeds predetermined threshold value based on the difference between initial and end number at least partly.
The test set that the order that detecting device can comprise series connection is arranged, the test set that the order of each series connection is arranged comprises the electrode at first pair of interval to measure the impedance in microchannel, capture region comprises and is configured to material interactional with one of plurality of target material, capture region is positioned at the downstream of the electrode at first pair of interval, and the electrode at second pair of interval is arranged in the downstream of capture region to measure the impedance of microchannel.Determine the initial number of impedance determination at least one target substance at the electrode place at first pair of interval that at least one target substance can be included in each test set place and measures based on the electrode pair at interval, and based on the end number of impedance determination at least one target substance at the electrode place at second pair of interval measured the electrode place at interval at another, and determine at each test set place whether the level of at least one target substance of catching in captured region exceeds predetermined threshold value at least partly based on the difference between initial and end number.
Except as otherwise noted, all technology used herein and scientific terminology have as usually or tradition understand the identical meaning.As used herein, article " a(mono-) " and " an(mono-) " refer to the grammar object of one or more than one (that is, at least one) article.Such as, " element " is meant to an element or more than one element.As used herein, " about " when the measurable value of finger, such as, when amount, duration etc., refer to comprise occurrence ± 20% or ± 10%, ± the change of 5% or ± 0.1%, because when system described herein, apparatus and method, these changes are applicable.It is be only conveniently and briefly that the disclosure mentions scope in the whole text, should not be construed as the fixing restriction of the embodiment to system described herein, apparatus and method.Therefore, the description of scope should be considered to specifically disclose all possible sub-scope within the scope of this, and other numerical value individual.Such as, the description of scope, such as should be considered to specifically disclose sub-scope from 1 to 6, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., and the indivedual numbers within the scope of this, such as 1,2,2.7,3,4,5,5.3,6 and the increment of any whole and part therebetween.
According to the detailed description of following accompanying drawing, the disclosure other and further object, feature, aspect, and advantage will become better understood.
Accompanying drawing explanation
With reference to following accompanying drawing, these and other aspect will be described in detail now.
Figure 1A comprises microchannel and electrode to produce the schematic diagram of the device in magnetic field.
Figure 1B is through the magnetic field (black arrow) of ferromagnetic microfluidic device xsect and the COMSOL simulation of magnetic flux density magnitude.
Fig. 1 C is the figure of the force and moment along the calculating on the microballoon of micro-tunnel length.
Fig. 1 D is the figure of magnetic force and the magnetic moment calculated as the function of frequency.
Fig. 2 A is the figure of the example embodiment of the optionally possible microchannel configuration of the electrode spread comprising 100,150,200 and 300 μm of spacing.
Fig. 2 B is the figure of the example passage with multiple entrance and exit.
Fig. 3 A is the schematic diagram of the example multidimension electrode configuration comprising crossed electrode layer.
Fig. 3 B is the figure of the electrode of concentric circles pattern.
Fig. 4 A is the installation drawing being implemented continuous stream operation by microchannel.
Fig. 4 B-D is the zoomed-in view of the various piece of device shown in Fig. 4 A.
Fig. 5 is the schematic diagram of another example embodiment realizing the device of discontinuous separation by continuous stream.
Fig. 6 A-C illustrates the figure using continuous stream to carry out the device of three lock out operation.
Fig. 7 is the process flow diagram to being suspended in the plurality of target material suspended in ferrofluid and carrying out the example procedure of discontinuous separation.
Fig. 8 A is the figure describing cobalt ferrite nanoparticles Size Distribution in ferrofluid.
Fig. 8 B describes the AC magnetic susceptibility of ferrofluid and the figure of demagnetization curve.
Fig. 8 C describes viable count to the figure of citric acid salt concentration.
Fig. 9 A-B comprises the figure being presented at and depositing ferrofluid endoparticle behavior in the case of magnetic fields.
Figure 10 A-D comprises the curve map and diagram that are presented at and deposit ferrofluid endoparticle behavior in the case of magnetic fields.
Figure 11 A and 11B is the figure of the cell separation describing various target substance.
Figure 12 A-C comprises the curve map and diagram that are presented at and deposit ferrofluid endoparticle behavior in the case of magnetic fields.
Figure 13 is the schematic diagram of microfluidic devices, and it comprises electrode group, and it is configured to produce magnetic field and the target substance of sample is focused on basic single file stream.
Figure 14 is the schematic diagram of the part being configured to another example microfluidic devices that target substance can be focused on alternately gap.
Figure 15 is the schematic diagram of the part of another example embodiment of microfluidic devices.
Figure 16 is the process flow diagram of the example procedure concentrating target substance in microchannel.
Figure 17 is the schematic diagram of the part of another example embodiment of microfluidic devices.
Figure 18 comprises the schematic diagram of capacitance detector and describes the figure of capacitance variations of bacterium by causing near detecting device face.
Figure 19 is the process flow diagram of the example procedure detecting at least one target substance in sample.
Figure 20 is the schematic diagram of general-purpose computing system.
Quotation mark similar in each figure represents similar element.
Describe in detail
System, method, device, product are disclosed herein, with various embodiment, comprise device (such as, plurality of target material for separating of being suspended in biocompatible ferrofluid), it comprises microchannel, this microchannel comprises at least one sample inlet and at least one outlet, and microchannel has the passage length extended between at least one sample inlet and at least one outlet.Microchannel is configured to accept continuous print sample stream substantially from least one sample inlet, and is configured to make sample flow at least one outlet along passage length.Sample comprises plurality of target material and biocompatible ferrofluid.Device also comprises multiple electrodes of the contiguous microchannel in position, multiple electrode is configured to when electric current is applied to multiple electrode, along the magnetic field model produced at least partially of microchannel length, magnetic field model is configured to when sample stream is along the advancing at least partly of microchannel, cause at least two kinds of plurality of target material in sample stream separated.
Also disclose the device concentrating at least one target substance be suspended in biocompatible ferrofluid.Device comprises microchannel, be configured to the sample accepting to comprise at least one target substance and biocompatible ferrofluid, accept at least one target substance in sample and be substantially concentrated in the inlet flow with correlated inputs width, with at least two electrodes of the contiguous microchannel in position, at least two electrodes are configured to, when controlled electric current puts at least two electrodes, in the sample comprising ferrofluid, produce controlled magnetic force.The controlled magnetic force produced causes at least one target substance to focus in produced stream, and its width is narrower than the relevant input width of inlet flow.
Further disclose system, device, method, and embodiment, comprise the device detecting at least one target substance in sample, device comprises microchannel, its be configured to accept to comprise at least one target substance and wherein at least one target substance be suspended in the sample of biocompatible ferrofluid wherein, the detecting device of at least one target substance in working sample, and at least two electrodes of the contiguous microchannel in position.At least two electrodes are configured to, when controlled at least one electric current is applied at least two electrodes, in the sample comprising ferrofluid, produce controlled magnetic force, and the controlled magnetic force produced causes at least one target substance guiding detecting device.
In general, system described herein, apparatus and method ferrofluid used is the colloid admixture of the magnetic-particle of nano-scale, and such as vectolite, covering surfaces activating agent, is suspended in the mounting medium compatible with surfactant material.Such as, the example reaction producing magnetic-particle is as follows:
2?FeCl3?+?FeCl2?+?8?NH3?+?4H2O?Fe3O4?+?8?NH4Cl。
The suspending liquid of the magnetic iron ore of 10 volume % has the saturation magnetization of about 560G.The magnetization of each single domain particle is 10 μ s magnitudes to the responsive time constant of highfield.High magnetic field gradients can be used for detent rail magnetic fluid.Under this highfield existent condition, " spike " and other significant features may appear at ferrofluid surface.
In some embodiments, particle diameter range can from about 1nm to about 100nm, and the increment of any whole or part therebetween.Such as, and without any restrictions, and particle diameter range can be 1-10nm, 1-20nm, 5-50nm or 10-100nm.In some embodiments, particle diameter average out to is about 10nm.Volume fraction can from 0.1% to about 10%, and the increment of any whole or part therebetween.
In some embodiments, ferrofluid described herein is biocompatible, and living cells can be made to maintain several hours, and physical property or continuation are not degenerated, and can extend the detection of target sample.Biocompatible ferrofluid goes for the cell type and/or the shape that maintain any work, such as any animal or plant tissue cell type, any microorganism, or its any combination.Ferrofluid can also be suitable for suspending the particle of any type and the particle of any size or shape, or cluster of particle or clump, no matter live or non-live.
Citrate is effective surfactant in ferrofluid, and its major part in cell culture is also compatible.Therefore, in some embodiments, citrate is used for stabilization ferrofluid and provides making cell survival ionic medium wherein.In this context, determine that new is wish with citric acid salt concentration that is optimum because very little or too many citrate can cause particle aggregation and precipitation in ferrofluid.Further, the survival of ferrofluid inner cell depends on to have enough ionic species to control osmotic pressure on cell to promote the continuation of cell.In an example embodiment, in the ferrofluid of the soliquid causing magnetic nanoparticle stable, citric acid salt concentration is about 40mM.And comparatively homocitric acid salinity may start to reduce ferrofluid stability gradually, the concentration of citrate can any position between 5-200mM, and any whole or Partial Incremental therebetween, depends on the characteristic sum type of ferrofluid used.(describe in Fig. 8 C) in example embodiment, further define and obtain pH about 7.4 with citric acid is stable, first cause the least concentration of the citrate in the most cells survival of several hours periods to be about 40mM.Under this ion concentration, cell has vigor and ferrofluid is stablized.Therefore, in some embodiments, the citric acid salt concentration of about 40mM can be used as effective biocompatible ferrofluid.As expectedly, and depend on the type of used ferrofluid herein, ferrofluid described herein can also be stabilized in following pH scope: about 2-11, and any whole or Partial Incremental therebetween.In some embodiments, ferrofluid is biocompatible, thus cell can survive in ferrofluid at least about 1 hour, about 2 hour, about 3 hours, about 5 hours, about 10 hours, high to about 24 hours or even longer.In some embodiments, biocompatible ferrofluid used comprises the biocompatible ferrofluid of design, there is the ionic strength of about 150mM, and this biocompatible ferrofluid is configured to isotonic eukaryotic such as people cell, the people cell etc. alive with being suitable for maintenance.
1. piece-rate system and device
As described herein, provide based on flowing molten iron body dynamics for the unmarked operation of bio-compatible ferrofluid target substance (such as, cell and microorganism) and the micro-fluidic system be separated.In some embodiments, system comprise as in microchannel around the ferrofluid based on water of the even magnetic environment of cell or other particle.Ferrofluid inner cell and other non-magnetic particle be similar to the mode of electron aperture in semiconductor serve as " magnetic space " (Kashevsky, 1997,
phys Fluids9:1811-1818).The outside magnetic field gradient applied can attract magnetic nanoparticle, and this causes non-magnetic particles or cell effectively to be pushed open (Rosensweig RE (1997)
ferrohydrodynamics(Dover, New York); Odenbach S (2002)
ferrofluids:Magnetically Controllable Fluids and TheirApplications(Springer, New York)).Recently, this principle be applied to catching non magnetic microballon in the microchannel of filling ferrofluid between thin magnetic film island (Yellen et al., 2005,
prot Natl Acad Sci USA102:8860-8864).In contrast, system of the present disclosure, apparatus and method comprise such as, microfluidic devices with electrode (can be optionally Integrated electrode, integrated copper electrode), and its transmission current produces with local can design/configurable magnetic field gradient.Appendix A below and B provide the mathematical description from magnetic field to microchannel (sample comprising target substance to be processed is flowed by it) and the analysis that cause behavior and the interactive relation occurred owing to applying.
Figure 1A-1D explains the example implementations of iron microfluidic devices and particle manipulation platform.Figure 1A comprises microchannel 110 and electrode 120(below draws not in scale) the schematic diagram of device 100.In some embodiments, in some embodiments, (such as, two output channels from amplifier provide the sinusoidal current of relative to each other PGC demodulation 90 °
i 1 with
i 2 ).Adjacent electrode on base material arranges in a certain way/connects, to support the travelling-magnetic-field in microchannel with orthogonal manner transport current.Non-magnetic microspheres in the magnetic field gradient promotion iron microchannel produced or cell rise and enter interelectrode gap.Traveling-wave field also causes cell along the rotary-top of passage and rolling, causes along this passage length with the frequency continuous translation higher than threshold value.The particle movement caused can with upright microscope 130 from top view to, and, such as use CCD camera with 18 frame-grabs per second, for further analysis.Iron microfluidic devices and further describing of particle manipulation platform are provided in, such as, on Dec 7th, 2010 submits to, PCT application PCT/US10/59270, the U.S.Provisional Serial 61/267 that exercise question was submitted to for " n cell undertaken by biocompatible ferrofluid is operated and sorting " and on Dec 7th, 2009, on October 28th, 163 and 2010 submit to U.S.Provisional Serial 61/407, in 738, its all the elements this by reference entirety be incorporated to.
Continue with reference to Figure 1A, electrode 120 can be arranged in electrode layer, and the contiguous microchannel 110 in position, can be positioned at above base material, the insulated metal substrate 150 of such as standard.Such as, bag can be used by the aluminium base of insulating polymer, and it can dispel the heat efficiently, and the AC electric current of such as 10A at the most can be made further to be conducted by electrode at lower voltages.In an example embodiment, single electrode layer can be used, described by Figure 1A.In some embodiments, multi-electrode layer be used for providing multidimensional control (with thus the multidimensional that can realize magnetic field control, described magnetic field can be produced and the sample put in microchannel with the target substance in operation and sample separation).Such as, Fig. 3 A describes the example of the multidimension electrode configuration comprising crossed electrode layer.
In given electrode layer, electrode can be about 30 μm high, about 300 μm wide and about 2cm length.In alternative embodiments, can be high at about 5-100 μm to the electrode in given layer, about 0.01-1mm is wide, and any range that about 0.1-10cm is long, and any whole or Partial Incremental therebetween.Therefore, will be appreciated that, the size of electrode used therein does not limit, and can change between multiple electrode layer.Further, electrode can comprise any shape, curvature or pattern, and can comprise variable gap size between electrode.Such as, Fig. 2 A describes the electrode spread at 100,150,200 and 300 μm of intervals in passage area.As described in, in some embodiments, multilayer orthogonal pattern can be used, such as, as described in Fig. 3 A.In an example embodiment, the concentric electrode pattern as described in Fig. 3 B can be used, with make row wave energy more effectively moving particles or cell enter round relevant range, leave round relevant range or circle relevant range different piece IT they.In some embodiments, electrode can have " wavy " (bending), or is generally bending shape, to introduce interference force and moment to neighbouring particle or cell.These wavy regions can be in fact even, uneven, random and/or periodic, and can be dispersed throughout electrode.It should be noted that, in a given electrode layer, the shape of electrode, interval and pattern may change, and can change at multiple interlayer further, make shape, size, any combination of interval and pattern can occur at electrode layer with across multilayer, to produce the magnetic field of the hope with desired attribute, (these attributes are controlled further, or configurable, such as, by controlling the electric current be applied on electrode).
In some embodiments, electrode can comprise any suitable conductive material, such as copper.In some embodiments, electrode can be made by the layers of copper of the printed circuit board (on insulated metal substrate) of photoresist mask wet etching thermal envelope.It is noted that any etching type or other suitable method for making may be used for producing electrode.
Passage (microchannel 110 such as shown in Figure 1A) can comprise at least one entrance and at least one outlet, and can be configured to make the length of passage by the vicinity of electrode at least partially, and can thus cross electrode at least partially.Such as, do in some embodiments, microchannel can rotate about 90 degree, thus the electrode of device is substantially parallel with its length.In some other embodiments, passage, with the angle of any whole and increment of part between about 0-90 degree and therebetween, crosses electrode.In further example embodiment, passage crosses electrode with basic straight line.In other other example embodiment, passage crosses electrode with arc, bending or usual irregular pattern.
In some embodiments, microchannel scope can be 20-100 μm high, 1-3 mm is wide and 2-3 cm is long, and any whole or Partial Incremental therebetween.Other size value can be used for passage.In some embodiments, passage can comprise the pocket of any number and size in conduit wall, ridge, trough and/or inclined-plane, thus the particle of advancing in passage or cell can based on the conformational effect concentration of local of conduit wall profile or dispersions.The additional examples with the passage of multiple entrance and exit describes in fig. 2b.Passage can comprise any suitable material.Such as, in some embodiments, passage can pass through soft lithography stamp, from dimethyl silicone polymer (PDMS) preparation, and be connected with the thin PDMS insulation course of coated electrode (Mao et al., 2006,
nanotechnology17:3447).In some embodiments, channel height can be selected lower than local flowing molten iron body dynamics stream optimal value, to minimize its potential effect for particle moving.
Although do not need, before ferrofluid/mixture of microspheres is imported microfluidic devices, passage with 1% triton-X solution cleaning about 10 minutes, can adhere to the particle of PDMS wall to minimize.Will be appreciated that, alternatively, base material, insulation course and passage can comprise the material with similar characteristics and/or character separately.Therefore, in order to define electrode, device described herein and system can be built upon such as be characterized as the insulation layers of copper etched by the transparent mask of single low resolution cheap printed circuit board on.As described in, microchannel can be built by using the soft lithography of low resolution mould.In some embodiments, device makes does not need clean room, and thus, can make fast and at an easy rate.
In order to operate sample to carry out desired process (such as to sample, be separated at least two kinds of plurality of target material, concentrate target substance etc.), by apply to electrode (electrode 120 described in such as Figure 1A) from power supply controlled at least one electric current (and, in some embodiments, at least two kinds of electric currents) in passage, produce the magnetic field of advancing, to create the magnetic field model at least partially along microchannel length.The alternating current of many about 7A peak-peak amplitudes and the frequency from about 10Hz to 100kHz can be applied to, the maximum magnetic field strength of about 90 Oe in this corresponding ferrofluid to electrode.In some changes, the magnetic field intensity scope produced can be 1-200 Oe and any whole or increment partly therebetween.In an example, magnetic field produces by applying orthogonal alternating current to single-layer electrodes, to create the periodic magnetic field mode of advancing along micro-tunnel length.According to this configuration, device can create the magnetic field gradient of the time average power caused cell or particle, and the magnetized local of ferrofluid rotates, and it finally causes the moment on non-magnetic particle, as shown in Figure 1B.Figure 1B describes preset time instantaneously through the magnetic field (black arrow) of ferromagnetic microfluidic device xsect and the COMSOL simulation of magnetic flux density magnitude.More shallow arrow describes the magnetic field of in one-period every 30 °.Shown analog result is carried out under the input of 12-A peak-to-peak current under 1670 Hz frequencies.
When controlled at least one electric current is applied to electrode, two or more target substance (cell or particle), at least partly due to magnetic force, is pushed to channel roof by from electrode, at least part of due to magnetic moment there, and they start to rotate and rolling along its length.Thus device behavior can simulate the magnetic susceptibility of the frequency dependence of used specific iron magnetic fluid.For given particle size, its speed can depend on the local force and moment value along passage length, such as, as shown in Figure 1 C (be presented under 4.6kHz under the peak-peak input excitation of 7-A, along the figure of the force and moment of the calculating of micro-tunnel length on the microballoon of 6-μm of diameter).
At low frequencies, magnetic force component is dominated usually, promotes non-magnetic particles upward to channel roof (that is, the surface that the surface of adjacent electrode is relative), and enters interelectrode space.At high frequencies, the particulate of rolling can overcome the repulsion reduced that magnetic force causes, and moves along passage continuously, as shown in figure ip.Fig. 1 D is the figure (the input current amplitude for carrying out simulating is 7A peak-peak, supposes that for the sliding rate of all simulations be 1) of magnetic force and the magnetic moment calculated as frequency function for the same particle in channel roof between electrode.
Such as, the typical magnetic force that can be applied on the particle of a few micron diameter can in the magnitude of tens skin newton, and it is significantly greater than light tweezer typical magnetic force on micrometer-sized particles.In some embodiments, driving force can increase by applying larger exciting current.Such as, peak-peak input current (Mao L, Koser H (2006) the Toward ferrofluidics for p-TAS and lab on-a-chip applications. of the content that simple heat radiator at room temperature can keep passage 10-A at the most
nanotechnotogy17:34-47).
Therefore, can be based on by the attribute (intensity, frequency, phase place etc.) applying the magnetic field that at least one electric current produces to electrode (such as electrode 120), such as, the special character of the electric current applied by electrode (such as, apply amplitude, frequency, the phase place of electric current).In some embodiments, the attribute being applied at least one electric current of electrode can use controller to control, such as based on the controller of processor, the controller 160(such as, described in Figure 1A or some other calculation elements), it can be defined as the current properties producing and keep the needs needed for some needed for magnetic field.Such as, in some embodiments, controller can make current properties carry out dynamics adjustment (such as based on the condition detected in device, if determine the non magnetic target substance in sample, such as particle, cells etc. do not have as required or expection process, and such as they are not properly separated, then change magnetic field).
Extraly, and/or alternatively, in some embodiments, produce the attribute in magnetic field can also based on the configuration of microchannel (such as, for implementing its structure, the material of passage), the configuration of electrode (such as, the spatial relationship of the relative microchannel of their layout, electrode, for implementing the material etc. of electrode), and other factors.Therefore, in order to control produced magnetic field to control and to regulate the separation function of such as device 100, one or more elements of component devices can be controlled or be operated.Such as, as as described in, in some embodiments, be applied to sample (to be provided by power supply by controllably applying at least one electric current to electrode (electrode 120 of such as Figure 1A) with the magnetic field making in sample in plurality of target material at least two kinds of energy separated, its can with tripping device, such as device 100 connects) control.In some embodiments, the electrode to device 100 controllably applies at least one electric current and can comprise and control/arrange power supply, thus power supply applies at least one electric current with selected separately amplitude, selected frequency and/or selected phase place.Under those environment, the separation of at least two kinds of target substances of sample can at least partly based on, apply one or more of selected separately amplitude, selected frequency and the selected phase place of electric current.
As described in, in some embodiments, to produce and the attribute putting on the magnetic field of sample can such as be determined according to the configuration of the electrode of the contiguous microchannel of the configuration of microchannel and/or position.Such as, referring again to Fig. 2 A, show the schematic diagram of the example embodiment of the optionally possible microchannel configuration of the electrode spread comprising 100,150,200 and 300 μm of spacing in passage.Fig. 2 B describes about between 0.17-0.19cm, has the passage arrangement of 4 entrances and 4 outlets.In another example, and referring again to Fig. 3 A and 3B, the schematic diagram describing the optional embodiment of electrode mode is shown.Particularly, Fig. 3 A describes substantially orthogonal multi-layered electrode pattern, and Fig. 3 B describes the electrode of concentric circles pattern.Each description in Fig. 2 and 3 configures thus provides another kind of method/mechanism control magnetic field properties, the target substance (passage 110 of such as Figure 1A) in the sample that it can be produced such as to be separated, concentrate, guide and/or operate in addition and driving is flowed in microchannel.
In some embodiments, tripping device, the device 100 of such as Figure 1A, can be configured to be separated the target substance be delivered to continuous stream in the sample of the microchannel of device.Show the figure of the continuous stream tripping device 200 of display section referring now to Fig. 4 A, Fig. 4 A, use and be configured to make the microchannel of target substance flowing in sample realize, the magnetic field produced by electrode is applied thereto.The device 100 that device 200 can describe in similar Figure 1A, or it can based on having, the different embodiments of such as different electrode configurations and/or different microchannel configuration.Fig. 4 A describes the sample 240 access to plant entrance that allows to suspend in ferrofluid 250 and by separation chamber, and the continuous stream device that the multiple outlets through being configured to the particle of catching special size are left.Particularly, device 200 comprises entrance (also referred to as the entrance stage) 210, separation chamber 220 and outlet (also referred to as the outlet stage) 230.In the example of Fig. 4 A, comprise two kinds of different target materials, namely the continuous stream sample of 2 μm of particles and 5 μm of particles, is imported by entrance 210.Such as, in some embodiments, sample can use at least one of the device of forcing pump, syringe pump, peristaltic pump, vacuum plant and/or other coupling entrance 210 any to import, and causes the entrance of sample 240 access to plant 200 and flows to separation chamber 220 and outlet 230.Extraly and/or alternatively, in some embodiments, continuous stream can be passed through gravity (difference in height such as, between entrance and exit pond) or be realized by capillary force.In Fig. 4 B, display comprises the zoomed-in view of the sample of two kinds of target substances.
Apply controlled electric current by the special electrode configuration of device 200, the magnetic field with association attributes (at least partly based on the controlled electric current applied by electrode) is produced, and causes target particles to be separated.Normally, being separated in when sample (comprising target substance) flows through separation chamber 220 of particle is carried out.As Fig. 4 A and Fig. 4 C(shows the zoomed-in view of separation chamber) as shown in, in the example described in Fig. 4 A and 4C, the magnetic field produced causes 2 μm of particles and 5 μm of particle separation, and the latter is captured in center clearance stream.Subsequently, and as shown in Fig. 4 A and 4D, 2 μm of outlet A flowing to the outlet stage 230, and 5 μm of grain flows are to the outlet B in outlet stage 230.Remaining sample flows to waste outlet C.The device 200 described in Fig. 4 A-D thus be suitable for separation and the sorting of two or more grain type, based on one or the combination of size, shape, elasticity, form etc.
With reference to Fig. 5, show the schematic diagram of another example embodiment realizing the device 300 of discontinuous separation by continuous stream.Device 300 comprises one or more entrance, such as, accepts the entrance 310 of ferrofluid and cell sample, and accepts two entrances 312 and 314 of ferrofluid.In some embodiments, can use the entrance of any number, each can be configured to accept will by the potpourri of the ferrofluid of device process and/or target substance, such as, by the controlled applying in the magnetic field that the microchannel of device leads.In the embodiment of Fig. 5, the material accepted by entrance can be accepted as the continuous stream provided by one or more pressure mechanisms, such as pressure mechanism 302, causes this continuous stream.As will be explained herein, provide the mechanism of continuous stream can comprise following one or more, such as, forcing pump, syringe pump, peristaltic pump, vacuum plant, equipment based on gravity, and/or capillary force.In some embodiments, the entrance stage can comprise centralized mechanism, and to be focused on by target substance in single stream, it is then by being applied to the magnetic field process of the microchannel of device 300.Centralized mechanism can be concentrated based on fluid mechanics and/or can according to concentrating (such as, using permanent magnet and/or another group electrode to produce the field being applied to sample near the entrance stage) based on magnetic.
As further illustrated in fig. 5, device 300 also comprises microchannel 320, and it can be similar to the passage 110 of Figure 1A, and thus can be various shape and configuration, and the one in multiple different materials can be built.Microchannel 320 is through multiple electrode 330(such as copper electrode, or the electrode of other type) vicinity is at least partially.Multiple electrode 330 can be configured to have size and other physical attribute, to promote to produce the controlled magnetic field (when controlled electric current is through electrode) with controlled attribute, for processing the sample of flowing in microchannel 320.Such as, in the embodiment of Fig. 5, the separation of the two or more target substance be suspended in ferrofluid can be carried out in the magnetic field produced, and is separated the result of multiple target substance as magnetic field, and the material of separation is directed at least one outlet pond.As shown in Figure 5, multiple electrode comprises 8 " wavy " electrodes, and it arranges that (such as, the various angles with relative microchannel 320) makes when controlled electric current is applied to multiple electrode 330, and the magnetic field with direction shown in arrow 350 is produced.Other attribute in the magnetic field produced can be controlled by least one electric current controlling to be applied to multiple electrode.In some embodiments, the electric current controlling to apply can use the controller 160 of controller as Figure 1A to carry out.Further, by using the difference configuration of electrode, the attribute in magnetic field can be adjusted further/be controlled, such as, to produce the magnetic field with different directions.The magnetic field that operative installations 300 produces causes the non magnetic target substance be suspended in ferrofluid to be directed to the region be substantially limited between electrode 332 and 334.In some embodiments, permanent magnet can be used to supplement or to substitute multiple electrode, to produce the magnetic field on the sample that is applied to and flows in microchannel.Such as permanent magnet may be used for applying the magnetic field gradient vertical with stream, to promote target substance, promotes the separation of target substance.
The magnetic field of advancing (such as, by the applying of at least one electric current, such as, having the applying of two kinds of electric currents of out of phase) produced causes producing magnetic force and torque, and it is applied to (such as cell, bacterium, other particle) on plurality of target material.This causes multiple target substance to be pushed (such as, using magnetic force upward to top with to interelectrode gap upper space) simultaneously and uses magnetic moment to cross the width (surface that the surface that such as, ionization electrode is nearest is relative) of passage along top rolling.In high frequency and small electrode gap, magnetic moment is dominated (cause cell advance the track of traverse gap), in low frequency and/or wide arc gap, and magnetic force leading (causing cellular retention to concentrate in each track).The magnetic field applied thus may be used for the discontinuous separation of realize target material, because in the device of clearance gap with increase, cell will finally be limited in respective track.Magnetic field causes the separation of target substance, and this is based on following one or more, such as, their respective size, they respective shape, their each own elasticity, their respective form and other characteristic of target substance.
When magnetic fields sample along microchannel length advance and close to the outlet stage time, many kinds of substance separated (such as, size, shape, form, elasticity etc. according to them), and be directed to respective separation outlet pond.Such as, in the embodiment that Fig. 5 describes, little target substance (such as, cellule or bacterium) drift about to the left side of device 300, such as, in the direction in outlet pond 344, larger target substance (such as leucocyte, red blood cell etc.) drifts about to different directions, such as, to outlet pond 342.Other material (such as, waste material) of sample can exit pond 340.In some embodiments, an outlet can only be used.
What the device 300 sending continuous sample stream realized particulate and cell continuous is high-throughoutly separated, operation and sorting.In some embodiments, target substance process/operation occur with outside apply to flow vertical.In the case, the transhipment of plurality of target material can be not coupling with the mechanism be separated.Flow velocity can be optimized for high flux, and cell separation area size can correspondingly be selected subsequently.Sheared from the sheath stream of side or by magnetic force by fluid dynamics, target substance (such as, cell) potpourri can focus on the single gap on adjacent electrode and between adjacent electrode.Continuation is separated and sorting cells by the magnetic field of advancing produced by electric current in electrode, when they drag to downstream with stream, crosses the width of separation chamber.
In some embodiments, can realize controlling by controlling excitation frequency, wherein target substance will be trapped in initial electrode gap, and target substance is by separated.Gradually changed by the gap width between adjacent electrode, also can realize other control of separation parameter.Larger gap makes it more easily catch and concentrates less particulate/cell.Such as, the gradient of adjacent segment size (such as, 100 μm, 200 μm with 300 μm) can be separated and then respectively concentrate 10 μm, 5 μm and 2 μm of microballoons, such as, as shown in figures 6 a-c, its display device, as the operation of device 300, uses continuous stream to carry out three separation.In some embodiments, similar method may be used for from blood, create the independent stream of leucocyte, red blood cell and blood platelet.This will make it within a few minutes, may obtain blood count results fast and exactly.Other target cell (tumour cell such as circulated) can be separated from remaining haemocyte, and this is by making their total hydrodynamic volume substantially be different from haemocyte to be reliably separated with microballoon (magnetic or nonmagnetic) mark them.Be different from common separation equipment, such as flow cytometer, typically at the cost of 100,000 dollars of levels, occupy whole bench top, and need many assemblies, comprise multiple laser instrument, pump, pipeline, reagent, fluorescent dye and those skilled in the art, discrete microchannel tripping device as herein described and system in minutes (instead of several hours) complete sample preparation, and in enough cheap packaging, it can be processed after process terminates.
With reference to Fig. 7, show the process flow diagram of the example procedure 400 plurality of target material be suspended in ferrofluid being carried out to discontinuous separation.Program 400 be included in microchannel as in Figure 1A, 4 and 5 describe microchannel entrance in accept 410 substantially continuous print comprise the sample stream of the multiple target substance be suspended in biocompatible ferrofluid.Biocompatible ferrofluid can be any ferrofluid described herein, is configured to maintain biological specimen (such as, human or animal's cell) and reaches the time period (such as a few minutes were by several hours) relatively extended.In order to the continuous print sample stream comprising multiple target fluid can be obtained, use following at least one, such as, forcing pump, syringe pump, peristaltic pump, vacuum plant, the structure/device of the pressure that gravity is assisted can be produced, and/or produce the device of capillary force, produce the pressure for providing continuous sample stream from external source.Also the device of other type can be used.
Accept sample at the entrance of the device based on microchannel, sample passes through 420 along microchannel.When sample flow is through the microchannel of device, the controlled electric current of at least one applies 430 such as, in the electrode (e.g., electrode, the electrode 330 described in Fig. 5) of multiple positions adjacent channel.Controlled at least one current arrangements is controllably produce magnetic field model along the passage length at least partially of microchannel, with cause at least two kinds of plurality of target material in sample separated.Especially, as described herein, controlled at least one electric current (and the configuration of selected electrode and/or the configuration of microchannel) makes the genus Properties Control in produced magnetic field make magnetic field be programmable or configurable.The magnetic field produced generally includes magnetic force and magnetic moment component, causes nonmagnetic substance (comprising target substance) to be pushed in ferromagnetic microchannel and rises and enter interelectrode gap.Field of advancing also causes cell along the rotary-top of passage and rolling, causes along this passage length with the frequency continuous translation higher than threshold value.The magnetic field produced causes different target substances in microchannel, have different spaces behavior and motion, and it depends on the size of target substance, shape, form, elasticity and further feature at least partly.Thus various target substance is separated, sorting or be distinguished from each other in addition, make the different target substance that is present in the sample of continuous flow identified.
Therefore, as described herein, can realize the pilot system based on stream, it merges biocompatible ferrofluid.In some embodiments, higher flux can realize by carrying out the separation of Biology-iron magnetic fluid, and the stream simultaneously flowed is continuously by the entrance of fresh target substance (such as, cell) introduction channel.In outlet, the pearl entered, cell, particle are sorted in different exit passageways.Therefrom, cell can be collected for detecting or directed outwards or inside (namely integrating) sensor.Such as, stream device allows the sample access to plant entrance that suspends in ferrofluid and by separation chamber, and leaves through the multiple outlets being applicable to the particle of catching special size.Such as, as described in Fig. 4 A, 2 μm of particles can flow into outlet A, and 5 μm of particles can flow into outlet B etc., and remaining sample can flow into outlet C.In some embodiments, do not need stream to guide cell.On the contrary, excitation can be used for guiding them.Further, such as, sensor can be integrated directly into along in the pocket on the side of circulation road.
Operation not only depends on cell size, also depends on cell shape and elasticity.Such as, bacterium and drepanocytic different size, shape and elasticity allow them to be separated from healthy blood cell.In some embodiments, particle separation can depend on size and frequency.In some embodiments, critical frequencies can also depend on electrode gap.Such as, larger microballoon can be first captured in less gap.By utilizing this phenomenon, sorting can be carried out based on particle or target size.Further, system can between the operation excitation of institute's selected frequency and another frequency alternately, and help to interrupt possible nanoparticle chains, it can be formed due to excitation.In some changes, " wavy " electrode can be used for preventing pearl or cell to be agglomerated into bulk when flows down channel." wavy " electrode introduces interference force and moment to smash reunion on pearl or cell, allows larger indivedual pearl or cell to arrange as the pearl on necklace.By periodically interrupting the chain of nano particle, the physical attribute of ferrofluid remains unchanged in time.Further, target cell can effectively, rapidly, and be concentrated in unmarked mode, catch, locate or orientation sensor surface simply.Such as separation method can based on size, shape, elasticity, form etc. by cell or grain type exit, or by increasing or reduce interelectrode spacing, catch cell or grain type by cell or grain type exit based on size, shape, form, elasticity and/or some other characteristic.
The program of separating at least one target cell (cell type) from sample that realizes is included in suspension cell in bio-compatible ferrofluid and forms sample, (these electrodes can the angle of microchannel 0-360o relatively be arranged to make sample pass through to cross multiple electrode, such as, when arranging with the angle of the longitudinal axis 90o of relative microchannel) microchannel, multiple electrode is substantially parallel with the length of microchannel.Program described herein may further include and applies electric current to create the magnetic field model along microchannel length to multiple electrode, and enters at least one output outlet based on the difference sorting cells of cell size, shape and flexible at least one.Separation can by effectively, rapidly, and concentrate in unmarked mode, catch, to locate or orientation sensor surface occurs simply.
Such as, in some embodiments, program described herein can also based on size isolation of target substances (such as cell and/or particle).This separation based on size can prove to have, such as, about 50% efficiency, about 60% efficiency, about 70% efficiency, about 80% efficiency, about 90% efficiency, about 92% efficiency, about 94% efficiency, about 96% efficiency, the efficiency of about 97%, the efficiency of about 98% and about 99% separation efficiency.Size resolution in separating treatment can be, such as, be less than about 10 μm, be less than about 9 μm, be less than about 8 μm, be less than about 7 μm, be less than about 6 μm, be less than about 5 μm, be less than about 4 μm, be less than about 3 μm, be less than about 2 μm, be less than about 1 μm, be less than about 0.5 μm, be less than about 0.1 μm and be less than about 10 nm.This separation can being less than about 2 minutes, be less than about 1 minute, be less than about 45 seconds, be less than about 30 seconds, be less than about 20 seconds and be less than in about 10 seconds and complete.
As further described herein, being separated of the continued operation of target substance and Shape-based interpolation (such as, cell, being separated of the red blood cell of such as living and sickle cell and/or bacterium) also can realize.This is outstanding proves that ferromagnetic microfluid is delivered to sensor array by quick separating with by target cell in raji cell assay Raji, significantly reduces incubation time and increases the ability of diagnostic sensitivity.
Micro-fluidic system described herein and device have the advantage of some uniquenesses, because they provide laminar flow platform to use under minute sample size.Systems/devices provides rapid diffusion and fast results further, can be portable, and can integrate other existing sensor (as will be hereafter described in further detail).Such as, system described herein, apparatus and method may be used for the sterilizing of the adult stem obtained from blood sample, for the background of soldier in fighting and seaman's wound healing and neomorph.System described herein, apparatus and method can also be used for low-level bacterial contamination in the blood that quick detection (such as, < 1 minute) contributes.Under Battlefield trauma injuries emergency, this can be useful especially.System described herein, device, method can also be used for the application of " looking for a needle in a haystack " formula needing the cell detecting extremely low concentration in blood, such as, search for the tumour cell circulated in blood.
experiments experiment example
In order to illustrate system described herein, the embodiment of apparatus and method and operation further, provide the following example.These embodiments are not interpreted as limiting system described herein, apparatus and method, and more should be interpreted as comprising any and all variants, and it becomes apparent as disclosed result provided herein.
Implementing hereafter in experiments experiment example, use co-precipitation program synthesis cobalt-ferrite nanometer particle, be finally merged into (Khalafalla SE, Reimers GW (1973) U.S. Patent number 3 in the ferrofluid (20% solids content) based on water, 764,540).By adding the potpourri of cobalt chloride hexahydrate (II) and iron chloride (III), cobalt ferrite nanoparticles is precipitated out from the 1M sodium hydroxide solution of boiling.Magnetic precipitation uses DI water cleaning twice.2 M nitric acid and 0.35 M ferric nitrate (III) solution (Massart, 1981, IEEE Trans Magn 17:1247-1248 is added in precipitation; Fischer etc., 2008, IEEE Int Conlon Nano/Micro Eng and Molecular Syst China, 907-910).Then potpourri stirs 20 minutes at 80 DEG C.Then salpeter solution is poured out gently and sediment magnet is fixed on appropriate location.Vectolite particle in precipitation was dispersed in DI water afterwards, and the ferrofluid obtained is dialysed 1 week to the sodium citrate of 40mM and citric acid solution (pH level 7.4).Between dialysis period, solution in every day, basis upgrades.The ferrofluid obtained has the viscosity of 1.5cP at 20 DEG C.
Implementing, hereafter in experiments experiment example, to use Tecnai 12 electron microscope (120 keV) from Philips to obtain transmission electron microscope (TEM) image.With C film covering copper/rhodium net (from Electron Microscopy Sciences) and be dipped into ethanol dilution ferrofluid sample.After obtaining TEM image, the particle size in image uses ImageJ software to characterize.From the probability density function matching of the magnetic nanoparticle core Size Distribution lognormal distribution that TEM image (counting about 200 particles) obtains, as follows
(equation 1)
Wherein
dthe stochastic variable describing core diameter, and
d 0 ln(is respectively with σ
d) mean value and standard deviation.
The AC magnetic susceptibility of the ferrofluid of frequency dependence, can obtain (Maiorov, 1979, Magnetohydrodynamics 15:135-139) by there is the mutual inductance change right with measuring solenoid when there is not ferrofluid.In this respect, pick-up loop (200 circles, mean diameter is 9.76 millimeters) concentrated in solenoidal field coil (340 circles, mean diameter is 13.34 millimeters), and the mutual inductance of two coils is shown (E4980A) to characterize by the LCR from Agilent.Ferrofluid sample is imported in two groups of coils in the plastic injector of 1cc.Symmetry in arranging ensures that the parallel field line of force can carry out mutual inductance analytical calculation according to the data recorded in pick-up loop position, and finally carries out analytical calculation to AC magnetic susceptibility.
Magnetization relaxation equation, supposes do not have fluid to move or convection current (Rosensweig RE (1997) Ferrohydrodynamics (Dover:New York)), is
(equation 2)
Wherein ω is the local vorticity in ferrofluid,
χ 0be the DC magnetic susceptibility value of ferrofluid, and τ is the magnetic relaxation time constant that magnetic nanoparticle is relevant.Cylinder arranges interior uniform magnetic field and causes symmetry, and it makes vorticity (and the Section 2 therefore in above-mentioned equation) negligible in measurement space.Magnetic relaxation time constant represents the combination of two physics relaxation processes.Magnetic core as fruit granule is enough little, their magnetic moment will rotate nano particle (N é el relaxation) (Rosensweig RE (1997) Ferrohydrodynamics (the Dover:New York)) of the inside simply, and characteristic time constant by
(equation 3)
Provide, wherein
f 0that precession frequency is (usually 10
8-10
12in the scope of hertz),
k a magnetic anisotropy energy density,
v core the core volume of nano particle, and
k b theat energy.The particle with larger core will have higher magnetic anisotropy energy, cause the magnetic moment that in-core is fixing, and particle general itself in the solution with externally-applied magnetic field rotating Vortex (Blang's relaxation), characteristic time constant by
(equation 4)
Provide.
Herein
ηthe dynamic viscosity of fluid,
k b boltzmann constant,
tabsolute temperature (Kelvin), and
d hyd be the Hydrodynamic diameter of particle, comprise its surfactant layer.The leading position of relaxation process is accounted for faster in these two kinds of mechanism.Vectolite has high magnetic anisotropy energy density (to bulk material at 1.8x10
5to 3.0x10
5j/m
3, and for nano particle up to 3.15 × 10
6j/m
3(people such as Tung, 2003,
j Appl Phys93:7486-7488)), and based on the ferrofluid relaxation of this material mainly by the particle rotation (Blang mechanism) higher than the critical nanoparticle size of about 5nm diameter.Because the most of nano particle observed in TEM picture is greater than this critical dimension, only Blang's time constant is considered in the measurement of AC magnetic susceptibility explaining us.
When not having vorticity, the sinusoidal steady-state solutions of equation 2 produces the concept of effective susceptibility, and it is as the function of frequency, describe ferrofluid the magnetization and apply between the amplitude in magnetic field and phase place relation:
(equation 5)
(Debye?PJW?(1929)?Polar?Molecules.?(Dover:?New?York))。Herein,
χ 0be the DC magnetic susceptibility value of ferrofluid, and τ is Blang's relaxation time that magnetic nanoparticle is relevant.
Ferrofluid is made up of the particle with Size Distribution (typically being lognormal distribution), and it also causes the relaxation time to distribute.Consider this point, the linear combination of all magnetic susceptibility spectrums that the particle size that overall AC magnetic susceptibility is described as existing in ferrofluid produces, the probability density function of the lognormal distribution of being correlated with by given particle diameter
f (D hyd )metering:
(equation 6)
In nano particle, the amplitude of total magnetization intensity is directly proportional to its core volume, and it is also like this to indivedual contributions of magnetic susceptibility spectrum.Therefore, in equation 1 probability density function by
v 2 core weigh.Normalized factor A is provided by following formula
(equation 7).
Suppose hydrodynamic diameter lognormal distribution (equation 4-7), AC susceptibility data (example as shown in Figure 8 B) can use the sinusoidal steady-state solutions matching of magnetic relaxation equation.Meanwhile, again suppose the lognormal distribution of identical hydrodynamic diameter (and granule density is as free parameter), the relative shape (as shown in Fig. 8 B illustration) of DC magnetization data can use Langevin equation model.Matching simultaneously explains experimental result well, obtains the average hydrodynamic diameter of 72.5 nm.This value is much larger than the average core diameter of the nano particle obtained with TEM.Reasonably explain it is that, in balance, nano particle forms middle-sized gathering, the magnetic field applied during measuring as single unit response for one of this difference.Also dynamic light scattering experiment has been carried out to the sample of the dilution of same ferrofluid.Those results confirm that hydrodynamic diameter is much larger than core diameter, support explanation in this paper.
Typically, the concentration of the surfactant used is high enough to prevent colloidal stability from degenerating (at least in some months) continuously.Therefore, possible particle aggregate is formed at one of the brief precipitate phase of ferrofluid synthetic schemes, and be speed up processing, it often relates to the use of permanent magnet.Surfactant added afterwards, can not smash the gathering formed.
In the further enforcement of hereafter experiments experiment example, the ZetaPALS equipment from Brookhaven Instruments Group is used to carry out dynamic light scattering experiment.Measure for these, multiple scattering is avoided in the dilution of ferrofluid DI water.Find that fluid dynamics particle diameter is 64.9nm.
In the extra enforcement of hereafter experiments experiment example, the particle manipulation device (device 100 such as, shown in Figure 1A) used in experiment comprises two parts: microchannel and copper electrode below.Electrode (30 μm high, 300 μm wide and 2cm length) can be made by the layers of copper of the printed circuit board (on insulated metal substrate) using photoresist mask wet etching thermal envelope.By applying to produce magnetic field of advancing in the channel with the alternating current of electrode single-layer orthogonal.Microchannel (20 μm are high to 100 μm, and 1mm to 3mm is wide and 2cm to 3cm long) can pass through soft lithography stamp, from dimethyl silicone polymer (PDMS) preparation, and be connected with the very thin PDMS insulation course of coated electrode (Mao et al., 2006,
nanotechnology17:34-47).Select far below the channel height of local flowing molten iron body dynamics stream optimal value, to minimize it for the potential effect of particle moving.In the separating experiment of tracer grain using submicron order, do not observe recognizable fluid dynamics stream.Insulated metal substrate can efficiently radiates heat, thus make at lower voltages can up to 10A by the AC electric current of electrode.Introduce ferrofluid/mixture of microspheres in microfluidic devices before, the passage Triton-X solution of 1% cleans about 10 minutes, to minimize the particle be attached on PDMS wall.
In the enforcement of experiments experiment example hereafter, from Duke Scientific (Fremont, CA, USA) obtain the green fluorescence polystyrene microsphere of different size (1.2 μm, 1.9 μm, 2.2 μm, 3.1 μm, 5.0 μm, 6.0 μm, 9.9 μm diameters).The microsphere diameter coefficient of variation is about 1%.The microballoon of these customized production has low-down factor of porosity and carries the charged group of minimum in its surface.Microballoon is suspended in deionization (DI) water, and is kept at 4 DEG C, until they are tested for ferrofluid.
In addition, in order to make cell visible in ferrofluid, haemocyte with green fluorescent membrane dyestuff PKH67(available from Sigma-Aldrich) dyeing.This dyestuff has excitation peak at 490nm, has emission peak (Horan et al., 1989, Nature 340:167 – 168) at 502nm.According to manufacturer scheme and have some amendments to carry out cell dyeing.
Common preparation scheme for carrying out hereafter experiments experiment example is as follows: get blood from contributor before experiment, be stored in 4 DEG C before dyeing.By about 1,000 ten thousand cell centrifugations, remove blood plasma subsequently.Then cell is suspended in not containing in 500 μ l RPMI 1640 nutrient culture media (available from Invitrogen, Carlsbad, CA, USA) of serum, and fully mixes with the cell removing any attachment and combination.The cell suspension obtained was 1000rpm centrifugal 5 minutes again.
Carefully blot supernatant, and bead is suspended in 500 μ l dilution C (being supplemented with staining kit).At once afterwards, 4 micromole PKH67 dyestuffs in dilution C are prepared in.Mix isopyknic dyestuff and cell solution.The cell suspension lucifuge obtained hatches 4 minutes.Stop staining reaction by adding equal-volume hyclone (FBS), and cell suspension hatches 1 minute further.Cell then centrifugal 5 minutes of 1200rpm to remove staining solution.They clean 3 times to remove the dyestuff in any remaining solution in containing the cell culture medium of 10%FBS.After cleaning completely, cell is suspended in nutrient culture media.The brightness fluorescent microscope of labeled cell is tested.Before mixing ferrofluid, Dulbecco phosphate buffered saline (PBS) (PBS) buffer solution for cleaning of cell containing 10%FBS of dyeing.
In addition, because citrate is effective surfactant in ferrofluid, and be biocompatible in most cases in cell chulture, therefore citrate is used as stable ferrofluid, and provides and make cells survival ionic medium wherein.In this context, determine that debita spissitudo is important because very little or too many citrate will cause particle aggregation and precipitation in ferrofluid.Further, the survival of ferrofluid inner cell depends on to have enough ionic species to control osmotic pressure on cell to promote continuation.Still the highest citric acid salt concentration in the ferrofluid of the stable soliquid of magnetic nanoparticle is caused to be confirmed as about 40mM.Higher citric acid salt concentration will start stabilization removal ferrofluid gradually.
Adopt trypan blue (available from Invitrogen) staining technique monitoring cell viability.Trypan blue is selectivity is blueness by dead cell dye, make living cells and dead cell can by dyestuff (the The Sigma-Aldrich Handbook of Stains distinguished, Dyes & Indicators, Green, F.J., ed., Aldrich Chemical Co. (Milwaukee, WI:1990), 721-722).According to the scheme of manufacturer, adding 10 μ l concentration to 90 μ l 0.4% Trypan Blue liquid is in every 1 milliliter of nutrient culture media 5 × 10
5the cell suspension of individual cell.After at room temperature hatching 5 minutes, the small sample taking from this potpourri is placed to hemocytometer with living cell counting.Determine first cause the least concentration of the citrate (obtaining pH 7.4 with citric acid is stable) of several hours periods most cells survival be 40mM(as shown in Figure 8 C).Under this ion concentration, cell has vigor and ferrofluid is stablized.Therefore, in all experiments relating to cell in the ferrofluid being suspended in us, use the citric acid salt concentration of 40mM.
embodiment 1: ferrofluid character and device characteristic
The ferrofluid of the high enrichment containing living cells is used always to be proved to be a challenge, because it needs well-designed colloid system.Maintain the maximally related ferrofluid parameter of living cells to comprise: pH, ionic strength and nano particle-surfactant combination, together with their entirety and relative concentration.
This respect that is combined in finding suitable nano particle-surfactant is very important: ferrofluid needs to stablize at pH 7.4, and colloid-stabilised sexual needs keep up to the ionic strength that can maintain living cells.Should also be noted that the Size Distribution of the nano particle in ferrofluid.If there is diameter to only have the nano particle of a few nanometer, they can pass through cell membrane, and cause direct cytotoxicity (Scherer etc., 2005, Brazilian J Phys 45:718-727).For this reason, system described herein, apparatus and method comprise the magnetic settling step in the synthesis of biocompatible ferrofluid, specifically to be stayed by minimum nano particle.
The commonsense method improving the biocompatibility of ferrofluid typically relates to thick polymeric layer, as glucosan forever covers the magnetic nanoparticle (people such as Bautista, 2004, Aranotechnology15:S154-S159), because surfactant molecule reduces toxicity by hindering the surface of directly contact inorganic nanoparticles.But such method causes the volume content of the magnetic nanoparticle in ferrofluid significantly to reduce, and the corresponding decline of its magnetic susceptibility.The magnetic susceptibility of higher ferrofluid is typically converted into particle manipulation faster, so ferrofluid of the present disclosure is by using short surfactant molecule optimization.
In some embodiments, the ferrofluid used comprises and suspending in water, and by the cobalt ferrite nanoparticles of citrate-stable.Average nanoparticle core diameter in the ferrofluid determined with transmission electron microscope (TEM) is found to be about 11.3 ± 4.4 nanometers (as shown in Figure 8 A, the figure of cobalt ferrite nanoparticles Size Distribution in the ferrofluid that display description is obtained by TEM, engineer's scale is 50nm).According to the synchronous matching to AC magnetic susceptibility and DC magnetization data, average hydrodynamic diameter is confirmed as about 72.5 nm.Especially, as shown in Figure 8 B, the AC magnetic susceptibility of display description ferrofluid and the figure of demagnetization curve, suppose the moderate particle aggregation matching of AC susceptibility data being shown to average hydrodynamic diameter 72.5 nm of lognormality Size Distribution.Particle aggregation to a certain degree in the soliquid that difference between the average hydrodynamic diameter observed in TEM image and independent core size shows ferrofluid.This discovery is also confirmed by dynamic light scattering measurement, and it obtains the average hydrodynamic diameter of about 64.9 nm on the ferrofluid sample of high dilution.But magnetic nanoparticle is compared still enough little with the microballoon of micron-scale with cell, to make ferrofluid approximate as continuous print magnetic medium.
In building-up process, determine that the best ion concentration in ferrofluid is about 40mM, to provide good balance between cell viability (being determined by Trypanblau test) and ferrofluid stability.Especially, with reference to figure 8C, provide the figure describing viable count and citric acid salt concentration.As shown in the figure, the citric acid salt concentration (stable to obtain pH 7.4 with citric acid) of 40mM is considered to the combination of cell viability and ferrofluid stability is best.Dotted line represents the cell count in original blood sample.Count the cell that 3 correspondences consume ≈ 1h in citrate solution.In the process of given experiment, cell keeps its vigor.The cell observing 75% maintains vigor, even if after suspending several hours in ferrofluid, makes to relate to living cells operation and can be extended with the test be separated.
Before cell manipulation experiment, ferromagnetic microfluidic device uses fluorescence polystyrene microsphere (Duke Scientific; Single dispersing is arranged, and diameter range is 1.2 to 9.9 μm).In order to understand excitation frequency and current amplitude to the impact of the behavior of the non-magnetic particles be dispersed in ferrofluid, the microballoon of different size is used to carry out series of experiments under different excitation frequencies and current amplitude.The microballoon mixing ferrofluid of a small amount of intended size (for minimum mean particle dia, every milliliter at the most 1.1 × 10
6individual microballoon), and add microchannel subsequently.Feeder connection and the clamping of outlet two ends, move to prevent transient state fluid.The microballoon of microchannel near top uses upright fluorescent microscope (Zeiss Axiolmager A1) and highly sensitive video camera (Retiga2000R) (adopting StreamPix software) from upper surface imaging.At MATLAB(MathWorks) in carry out offline image analysis by light stream program.This program can being less than automatic pursuit path in (1) minute in the visual field, and determine the size of thousands of independent microballoons.
In these experiments, the dynamics of two class particles is observed.When low frequency, in-between the electrodes, the repulsive force that wherein magnetic field gradient causes forms Local Minimum in microballoon location.With reference to Fig. 9 A, higher than critical value
f c frequency, cause microballoon along channel roof length continuous translation.This threshold frequency depends on particle size and electrode separation, but must not depend on the amplitude (Fig. 9 B) of input current.The average velocity of intended size microballoon depends on the position of the amplitude of excitation frequency, electric current lower electrodes relative to them.
Especially, Fig. 9 A and 9B shows the function of particle speed as incoming frequency and current amplitude.In figure 9 a, under middle figure is presented at the input current amplitude (peak-peak) of 7-A, under two different frequencies, the space distribution of the instantaneous average x speed of diameter 6pm particle.Due to the repulsive force from magnetic field gradient, particulate slows down in-between the electrodes or stops completely.The equilibrium point (i.e. particle capture) that zero passage negative slope is corresponding stable.The figure at Fig. 9 A top shows, and at 10 Hz, particle trajectories stops in-between the electrodes, causes and catches.Figure bottom Fig. 9 A shows, and 4,640 Hz, particle is continuous moving in passage length.This magnetic moment carrying out the local rotational component of ripple voluntarily dominates the region exceeding repulsive force.The stain of every bar trailing end represents the final stopping place of particle.Fig. 9 B show higher than threshold frequency (
f c ) after, the microballoon of 6 μm rolls continuously along top passageway surface and is not captured.
The dependent particle separation of Figure 10 A-10D display frequency.In the experiment of various microsphere diameter, find to increase with particle size, threshold frequency monotone increasing, show by the potentiality of excitation frequency control for the particle separation based on size.Figure 10 A display threshold frequency (
f c ) particle size dependence.The particle of different-diameter is discrete
f c value enables the separation being undertaken based on size by being transferred to suitable frequency.This phenomenon can be explained by the reasoning of simple hydrodynamic force mechanics.Magnetic force and magnetic moment with particle volume (
r 3 ) weigh, resist R and the relative magnetic moment that particle is rolled of the hydrodynamic force mechanical resistance relative magnetic of linear particle motion
r 2 weigh.Therefore, the linear particle speed that magnetic force causes separately depends on
r 2 , and magnetic moment causes weighs with R.This observation shows, magnetic moment is relatively more remarkable on the impact of less particle, and explains why less particulate and can overcome the repulsion that magnetic force catches, and spreads continuously in passage under more low frequency.Block curve shown in Figure 10 A represents the analog result of threshold frequency and explains the average globule wall gap of nanometer (1 nanometer) well and be applied to the data of non-slip condition of rotation of microballoon.
Figure 10 B show 2.2 with the average velocity (also referred to as operating speed) of microballoon (mixing in the ratio of 8:1 in identical ferrofluid) under the excitation frequency of 10Hz to 100kHz of 9.9 μm.As shown in Figure 10 B, 2.2-with 9.9-μm of particle can be separated under 400 Hz.For wide frequency range, less particle shifts continuously, and larger particle is captured in-between the electrodes.At this in specific and other experiment, particle/cell mixture is finally divided into two groups, and such as, those relative those be captured are removed from passage.Hypothetical target particle/cell is that those are intended for and catch, and capture rate can be defined as the ratio of the number in its corresponding original mixture of number of the target part in captured group.Equally, separation efficiency is defined as the ratio of the number of non-targeted part in the group that the is eliminated number corresponding in original mixture to them.But particle/cell purity is only catch the number of group internal object cell and the ratio of total cellular score in this group.Under the excitation frequency of 400 Hz, the microballoon (167 in 173) of 9.9-μm of 96.5% is captured in 10 seconds, and 2.2-μm of particle (1, in 294 1,285) continue shift along passage and be eliminated out form (45s) and not captured (as illustrated in figs. loc and 10d), there is the separation efficiency of 99.3%.Catching particle purity in group is 94.9%(176 total 167 targets be captured in particle).The most minimicrosphere failing to remove from passage is bonded at position random on dimethyl silicone polymer (PDMS) wall, instead of captured between electrode.Prepared by passage better, and separation efficiency and particle purity can be higher.
Figure 10 C is the fluorescence microscopy images containing being just randomly dispersed within a section of the microchannel of 2.2 in passage and 9.9-μm of microballoon before excitation.Perpendicular line represents boundary electrode.Figure 10 D is the passage snapshot opened in excitation (6A peak-peak, 400Hz) from position identical shown in Figure 10 C latter 45 seconds.9.9-μm particle quick position in spacing between nearest electrode, and the microballoon of the 2.2-of 97% μm consecutive row and then be not captured from right to left.Less microballoon nearly all in the visual field in Figure 10 D enters from the right as newly a collection of.
Figure 11 A and Figure 11 B describes the cell separation of bacterium and haemocyte.Figure 11 A be described in comprise E. coli bacteria (
e. coli) and erythrocytic sample under 200Hz the space distribution of x speed.Under this frequency, most of red blood cell is captured (showing by their zero local velocity) in-between the electrodes, and Escherichia coli can slowly but be moved continuously through this region.As described herein, the fluctuation in RBC number certificate is statistical property.Figure 11 B describes sickle cell and is separated.Sickle cell, has the shape of elongation and the elasticity of change compared with normocyte, captured and concentrated in-between the electrodes, and healthy cell still can at microchannel Inner eycle under 300Hz.The electrode separation of the device in Figure 11 A is different from Figure 11 B, causes in each passage erythrocytic
f c different.
Figure 12 A describes the average velocity of particulate, is normalized, is described as the function of excitation frequency by the maximal value of square relative 12A of current amplitude (peak-peak).Square being directly proportional, until about 7A of particle speed and electric current.Under Figure 12 B is described in 6A peak-peak, the mean speed relative frequency that 9.9pm microballoon is advanced at the electrode of different spacing.Less spacing causes higher particle speed and less threshold frequency.Figure 12 C describes the concept sketches of the particle sorter on the basis of the effect observed in fig. 12 c.Under given excitation frequency, less spacing catches larger particle, and allows less particle pass through.Finally, even minimum particle also can be trapped in larger gap.Therefore, suppose that particle moves from left to right, and the passage of described electrode is acellular at first.
Therefore, movement of particles is confirmed as depending on electrode separation, and less spacing causes microballoon faster to be advanced and the minimizing (see Figure 12 B) of threshold frequency.This phenomenon may be used for device, it is characterized in that the electrode zone with different gap, to use same excitation frequency to be separated the granulate mixture had more than two different sizes.Electrode mode can also use the gap that increases gradually to create, with based on size separation particle (such as, as shown in figure 12 c).In this respect, observe the resolution that the medium and small unevenness (manufacture causes) of actual electrode separation is partly determined to be separated, be defined as still can be minimum in the particle that efficiently (such as, > 90%) is separated size difference.The particle size of given certain limit, this isolation resolution is directly involved in the difference in corresponding threshold frequency.Under ideal conditions (that is, the electrode gap preferably controlled and very rare cell concentration), isolation resolution can be arbitrarily small.But around each uneven electrode gap, threshold frequency is tended to show slight localized variation.As shown in Figure 10 A, best threshold frequency non-linearly depends on particle radius.Therefore, between 9-μm and 10-μm of microballoon, 1-μm diversity ratio 1-μm of diameter and 2-μm of intergranular 1-μm difference are more easily differentiated (in electrode separation, having slightly random change).Finally, the resolution of the separation realized in experiment described herein is ≈ 1 μm for 2 μm or larger particle.
embodiment 2: current amplitude is on the impact of particle speed
In additional experiment, study and determine the dependence of particle manipulating speed as input current magnitude function.According to the calculating summarized, and suppose that ferrofluid keeps linear magnetic herein, square measurement of particle speed input current.As illustrated in fig. 12, this hypothesis starts to break through the peak-peak input current amplitude higher than 7A.
Electrode separation also changes (electrode width is fixed on 210 μm) to determine its impact on particle manipulation.Less electrode separation causes average grain speed and lower threshold frequency (as shown in Figure 12 B) faster.This observation can by pointing out that electrode separation is explained closer to the local magnetic field gradient reduced particulate generation magnetic force.Closer to electrode also more multi-energy is put into the fundamental frequency of the row ripple these particulates being produced to magnetic moment.Lower magnetic force and the magnetic moment of Geng Gao cause particulate faster to rotate and overall gait of march.They also cause lower (Tung LD, et al. (2003) the Magnetic properties of ultrafine cobalt ferrite particles. of threshold frequency
j Appl Phys93:7486-7488).This observes the idea supporting to use granule separator further, and the spacing wherein between electrode can increase to catch more and more less particle or cell (as indicated in fig. 12 c) gradually.
embodiment 3: the separation of living cells
Based on the physical behavio(u)r of ferromagnetic miniflow platform, undertaken operating and separating experiment by the human red blood cell of living and bacterium, as herein described for the ferromagnetic micro-fluidic system of biomedical applications and the effectiveness of device and practicality to prove.Red blood cell and E. coli bacteria [K12 bacterial strain (Blattner etc., 1997,
science277:1453-1474))] dyeed with green fluorescent marker and mix before being suspended in ferrofluid.The peak-to-peak current amplitude of the average velocity 6A of the cell in passage and bacterium is measured under the frequency from 10Hz to 100kHz.Find that the threshold frequency of cell and bacterium is respectively 215 and 77Hz.These
f c value, slightly lower than the polystyrene microsphere that size is suitable, may be the shape owing to complying with and the geometric combination of aspherical causing increasing along the difficulty of channel roof rolling.In addition, there is bacterium and the cell of the surface chemistry of its complexity, with the interaction of PDMS passage than naked microballoon stronger (causing more general cell to adhere to), show higher effective kinetic friction coefficient potential between cell and PDMS surface.Refer again to Figure 11 A, to show under the excitation frequency of 200 Hz cell and bacterium along the linear velocity of the space average of passage.Less Escherichia coli are moved (speed point in Figure 11 A does not have zero passage) continuously along passage, and finally leave form, and haemocyte is positioned (speed point reaches zero) between electrode.It should be noted that, the larger change observed in the RBC number certificate of Figure 11 A comes from the fluctuation of statistics: only have a small amount of red blood cell in form by given x position, and their non-spherical shape means, when it rolls passage in this position, each cell will be random angular orientation (with slightly different instantaneous velocity).Bacterium, although the different and aspherical of length, also has enough number (hundreds of by given x position) to obtain good average statistics.Finally, be presented on 7 in the sample visual field at first, the ≈ in 050 Escherichia coli 6,750 was eliminated (95.7% separation efficiency) in 45 seconds.Present at first 1,018 erythrocytic 954 are captured, the corresponding capture rate of 93.7% and the cell purity of 76.1%.
In different experiments, healthy red blood cell and those suffer from sicklemic cell separation, this is by utilizing the shape between them and flexible difference (as shown in Figure 11 B).The erythrocytic blood sample of health to falciform comprising ratio and be about 4:1 is added to ferrofluid and imports microchannel.At 300Hz, sickle cell is captured, and the haemocyte of health is eliminated continuously (fluctuation of each data centralization shown in Figure 11 B is statistical property) from passage.Comprising at first in the healthy red blood cell of ≈ 501 and 145 drepanocytic samples, 300 healthy cells are eliminated, and 109 sickle cells are captured.Hypothetical target removes sample from sickle cell, the separation efficiency (109 in 145 sickle cells are separated from healthy cell) of these numerals corresponding 75.2%, and the healthy cell purity of 89.3% (300 healthy cells and 36 sickle cells are eliminated).
The ferromagnetic microfluid of these examples proves is delivered to sensor array by quick separating with by target cell selectivity in raji cell assay Raji, significantly reduces incubation time and increases the purposes of diagnostic sensitivity.Although particulate also can be undertaken by built vertical technology (such as DEP and the method based on magnetic mark) with being separated with the operation of living cells in microfluidic devices, ferromagnetic microfluidic methods as herein described has many advantages compared with the conventional method.Such as, target cell can effectively, rapidly, and be concentrated in unmarked mode, catch, locate or orientation sensor surface simply.The biocompatible ferrofluid of the present disclosure cell that can make to invigorate blood circulation maintains several hours, and physical property is not degenerated, and allows the detection extending target sample.
With simple photodiode in conjunction with time, the ferromagnetic microfluidic separation of cell can provide fast, robotization and disposable blood measure, it in the concentration of such as inside counting in 1 minute and any target cell type of estimation (as bacterium or sickle cell), can not need microscope, pump or tediously long sample preparation steps.System as herein described, apparatus and method can also be used for selectivity and concentrate rare cell, such as, circulating tumor cell in blood sample, by utilizing difference (Lekka et al., 1999 of the Young modulus (Young's modulus) of target cell type
eur Biophys J28:312-316).The system as herein described applied in this way, apparatus and method can increase the detection sensitivity of existing raji cell assay Raji.
System as herein described, apparatus and method thus use cell manipulation and the separation platform of biocompatible ferrofluid in the microfluidic devices being included in low cost.Proved can be less than in one (1) minute realize high-efficient granule be separated.As an example, bacterium can be separated from the haemocyte of living, and sickle cell can be separated from healthy red blood.For the device based on stream, be separated and can be realized by the particle manipulation vertical with flow path direction.By changing geometry and the input excitation frequency of electrode, system as herein described, apparatus and method are applicable to particle and the cell of different size scope.Together with the control of the surface chemistry of microchannel, system as herein described, apparatus and method can be incorporated in chip lab sensor and diagnostic system with active region of being led by target cell.In this fashion, system as herein described, apparatus and method can reduce incubation time significantly and increase in existing sensor and diagnostic platform the actual detection sensitivity realized.
2. target substance is concentrated
As described in, go back descriptive system, device, method and other embodiment herein, comprise the device concentrated and be suspended at least one target substance in biocompatible ferrofluid, it comprises the microchannel being configured to sample and the biocompatible ferrofluid accepting to comprise at least one target substance, at least one target substance accepted in sample be substantially concentrated in the inlet flow with correlated inputs width.This device comprises at least two electrodes of the contiguous microchannel in position further, described at least two electrodes are configured to when described in controllable electric electric current is applied to during at least two electrodes, controlled magnetic force is produced in containing the sample of ferrofluid, the controllable magnetic produced causes at least one target substance to concentrate in obtained stream, and its width is narrower than the relevant input width of inlet flow.In some embodiments, the magnetic force produced can be controlled by the controlled applying of at least electric current to electrode (that is, with about the similar fashion described in the magnetic field controlled for making the discontinuous separation of plurality of target substance).
As described herein, the advantage of microchannel and electrode technology is that it concentrates the ability arranged in single file with concentrating cells in channel roof.Therefore, due to the electrode of potential tiny model, having this ability can realize high precision.Use from the magnetic field of external permanent magnet, also can realize concentrating to a certain degree.Cell is pushed to the top of passage, and concentrate them to make it be easy to detect, count and catch them.
As will be described in more detail below, there are at least three kinds of modes, Different electrodes geometry wherein can be used in ferromagnetic microfluidic devices to realize cell and concentrate: cell is shifted onto the space above the gap between them by least two parallel poles that (i) carries DC or AC electric current (relative phases with different) along the length of passage; (ii) at least two groups in the opposite direction carry the electrode in magnetic field of advancing, and cell can be made to roll to border often between group; (III) carries DC or AC electric current (at different relative phases, comprise those of the field that causes advancing), the array of the electrode that gap reduces gradually between adjacent, remains on cell in those gaps by promotion, until gap becomes enough little, become outstanding to make the effect of magnetic moment.First two method can be used for target cell stream to concentrate near detecting device/counter, and the desirable large-scale parallel for realizing inputting sample of the third method is concentrated and being separated subsequently.Specifically, in the third method, first target substance (such as, cell) can be concentrated in alternately catches in gap, and target cell will be split in adjacent segment subsequently.
The advantage using electrode and microchannel configuration to realize cell concentrated is as follows:
Because do not have fluid dynamics sheath stream, can realize very effective concentrating, the whole volume of device can be that input stream of cells is special.Therefore, use embodiment described herein to compare the existing method using fluid dynamics to concentrate and can realize more high flux.
By input stream of cells being focused on the electrode gap every, target substance (such as, cell or pathogen) can be separated to adjacent gap.This ability makes to achieve theatrical walking abreast in separation and sorting function, because the characteristic of this equipment is the many such electrode mode crossing over the arrangement of circulation road width.
When cell lines up single file at the near top of passage, they can be detected easily (by various mode, comprise, such as, light, magnetic, electrical impedance and capacitance measurement) and counting.By this way, the systems/devices realized can use as " nano cell counter ", can be separated millions of cells and count within a few minutes.Systems/devices can be used as the detecting device of pathogen, or may be used for the concentration helping other untargeted cells of sign (as CD4, red blood cell, blood platelet etc.).
With reference to Figure 13, the schematic diagram of display microfluidic devices 500, it comprises electrode group, and it is configured to produce magnetic field so that the target substance of sample is focused on basic single file stream.Device 500 comprises microchannel device 510 and electrode group 520.Microchannel 510 can be similar to Figure 1A, microchannel described in 4 and 5.Electrode group 520 comprises, and comprises two usual " wavy " electrodes substantially parallel to each other in the example embodiment of Figure 13.Produce magnetic field when applying controlled at least one electric current to electrode group, cause the target substance be suspended in ferrofluid be pushed in microchannel and concentrate.The controlled at least one electric current being applied to electrode can use the controller 160(of controller such as Figure 1A can be controller based on processor) control/regulate.
In some embodiments, described at least two electrodes (such as electrode 520) can comprise the structure with following at least one, such as, one or more substantially straight shape of at least two electrodes, one or more substantially corrugated shape of at least two electrodes, the substantially parallel layout of at least two electrodes, and/or the location of at least two electrodes taper substantially, wherein said at least two electrodes are close to each other gradually.
In some embodiments, electrode group be configured to produce magnetic field, when apply described at least one electric current thereon time, it is applied to target substance at the entrance area of microfluidic devices substantially.In these embodiments, multiple electrode group can be used to microfluidic devices, do in order to concentrate the sample comprising at least one target substance in the single-row layout flowing through microchannel for one group, and another electrode group, its fundamental purpose implements discontinuous separation (such as, continuous stream to based on ferrofluid) sample, so that two or more different target substance can be separated in the mode related to such as described in Fig. 1-7.
In some embodiments, concentrate on the magnetic force of microfluidic devices entrance, can cause cell to be sorted into fast define in microfluidic devices catch the gap passage of this device (such as, in).Therefore, this centralized function may be used for first concentrating target substance (such as, target cell) to alternately catching, and then target cell is separated to adjacent segment.
Therefore, with reference to Figure 14, display is configured to the schematic diagram of a part for another example microfluidic devices 600 that target substance can be focused on alternately gap.Device 600 comprises microchannel 610, and it can be similar to about the microchannel described in Figure 1A, 4,5 and 13.Device 600 comprises one group of electrode with multiple electrode 620 further, and it comprises at least two class geometric electrodes.In the example of Figure 14, one (definition pair of electrodes) of every two adjacent electrodes has the length of the entrance area 630 substantially extending to device, with thus definition set electrode 622.Adjacent with each this concentrated electrode 622 is shorter electrode 624.In some embodiments, can use pair of electrodes, it comprises concentrated electrode 622 and shorter electrode 624, and these arrangement of electrodes are the location of taper substantially relative to each other, thus they are close to each other.Multiple electrode 620(comprises concentrated electrode and shorter electrode) to light from microchannel there is wavy shape, it is through the entrance stage, and extends the exit region to device.In some embodiments, take the locus of usual waved configuration from multiple electrode 620, electrode extends with direction substantially parallel to each other usually.
According to the example geometry of electrode shown in Figure 14, when applying at least one electric current to multiple electrode 620, the geometry of the electrode around entrance area causes producing magnetic field, this causes target substance to be directed to multiple single file stream, and it concentrates on alternately catching in gap of electrode pair definition and (comprises shown in Figure 14 and catch gap 640).The similar mixtures of the target substance that device portal accepts (its each can comprise) is formed once the stream of flowing, the magnetic field acting on the multiple sample streams concentrated now causes different target substance separation to enter adjacent segment (namely, as the result of centralized operation, do not accept the gap of any target substance).Magnetic field (this place's electrode 620 is parallel substantially, and its operation is the to carry out discontinuous lock out operation) magnetic field compared around inlet region that flow channel area place is formed can have the attribute of different (or similar).The discontinuous lock out operation undertaken by the magnetic field of substantially parallel electrode 620 position is similar to the operation carried out about system, equipment and method described in Fig. 1-12.
Therefore, in some embodiments, device can comprise electrod-array, and at least some first arrangement of electrodes of array is the location becoming basic taper relative to adjacent electrode, makes described first electrode be configured to move closer to adjacent electrode.Electrod-array be configured to produce magnetic force cause the stream of obtained at least one target substance above adjacent electrode is right and between formed.Centralized operation rear electrode between each gap thus become empty gradually, but when carrying out lock out operation to flowing stream, empty gap is received in the target substance that at least one that flows in adjacent segment is separated from potpourri, and wherein said potpourri is concentrated in adjacent segment.
May be used for exercising centralized function from those other different geometric electrodes shown in Figure 13 and 14.
In some embodiments, the electrode for carrying out centralized operation can be configured at least two magnetic waves producing backward-travelling wave field, focuses on border immediate vicinity between at least two magnetic waves produced to cause at least one target substance.With reference to Figure 15, show the schematic diagram of the part of another example embodiment of microfluidic devices 700.Device 700 comprises microchannel 710, and it can be similar to about the microchannel described in Figure 1A, 4,5,13 and/or 14.In the embodiment of Figure 15, device 700 comprises one group of electrode with multiple electrode 720 further, has usual wavy shape, and has substantially parallel location relative to each other.The contiguous microchannel 710 in electrode 720 position.Such as, electrode 720 can be arranged in the electrode layer be arranged in outside microchannel.
When electrode to Figure 15 applies controlled at least one electric current, produce two reverse row field magnetic waves (being labeled as ripple 730 and 732).Near the border 740 that two magnetic waves of reverse capable field cause target substance in sample stream to be concentrated between two magnetic waves produced (being described as dotted line).
Other electrode configuration may be used for produce in the sample to which concentrate target substance magnetic field (with thus produce magnetic force).In some embodiments, be configured to produce the controlled AC electric current that can conduct the controlling attribute with the centralized function desired by being defined as implementing at least two electrodes in the magnetic field of carrying out centralized function.In some embodiments, at least two these electrodes can conduct direct current electric current.In some embodiments, single electrode can wrap up self with formed two (or more) parallel arms, it defines multiple electrode part for generation of centered magnetic field.In some embodiments, one or more permanent magnet group may be used for supplement or alternative electrode group to help the enforcement of centralized function.
With reference to Figure 16, show the process flow diagram of the example procedure 800 concentrating target substance in microchannel.Program 800 comprises the sample that acceptance 810 comprises at least one target substance be suspended in biocompatible ferrofluid, and at least one target substance in the sample of acceptance is concentrated in the inlet flow with relevant input width substantially.
After accepting sample, at least one electric current (such as, there is the AC electric current of controlled frequency, phase place and/or amplitude, DC electric current etc.) controllably apply on the electrode of the contiguous microfluid in 820 at least two positions, to produce controlled magnetic force in the ferrofluid passage comprising sample.Magnetic force causes at least one target substance to focus in produced stream, and its width is narrower than the relevant input width of inlet flow.In some embodiments, at least two electrodes are configured to conduct the electric current that controllably provides and produce controlled magnetic force, according to, the attribute (such as its structure or shape) of the such as attribute of applied electric current, the attribute of microchannel and/or at least two electrodes.
3. lead target substance the magnetic force of detecting device
As mentioned previously, there is also described herein device, system, method and embodiment, comprise the device detecting at least one target substance in sample, described device comprises the microchannel being configured to the sample accepting the biocompatible ferrofluid comprising at least one target substance and wherein suspension at least one target substance, determine the detecting device of at least one target substance in sample, with at least two electrodes of the contiguous microchannel in position, described electrode is configured in the sample comprising ferrofluid, produce controlled magnetic force when controlled at least one electric current is applied at least two electrodes.The controlled magnetic force produced causes at least one target substance guiding detecting device.Because the effect and the quality that detect target substance depend on the vertical range of target substance to electrode at least in part, if vertical range can not be controlled, then dissimilar target substance can not reliably be distinguished.Correspondingly, by target substance being shifted onto top (neighbouring can arrange testing mechanism), as the result of target substance to the vertical range of detecting device, hinder the challenge successfully detecting target substance can be overcome to a certain extent by control.
In some embodiments, detection scheme as herein described relates to the direct impedance bioelectrical measurement by pair of parallel plane electrode, and/or capacitance measurement.About impedance measurement detecting device, the cell size scope that needs are counted and detect determines the spacing between these electrodes.Typically, this spacing should be equivalent to or slightly larger than cell dia.For haemocyte, spacing can be 10-20 micron.For Bacteria Detection, spacing can be 5-10 micron.
Conductivity by measuring the buffer solution flowed in microchannel between two electrodes through a small amount of electric current is comprised to the existence counting of target substance in sample and the various technique/method of detection.Any fast peak in this electric current (AC or DC) will show the of short duration change of the local conductivity of medium-most possibly caused by the process of cell, and its electric conductivity is different from medium.When gap between sensing electrode is suitable with cell size, the interference of electric field patterns is in-between the electrodes maximum, and electric signal to noise ratio (S/N ratio) is best.
In some embodiments, can be undertaken by measurement electric capacity the detection of the cell existed in ferrofluid solution and counting.The electrode lay-out of capacitive sensor is identical with impedance method with cell detection mechanism: pair of electrodes, has the gap that width is suitable with interested cell dia.The electric capacity measured is little typically, and general high-frequency AC excitation (typically, >1 MHz) of using of its measurement carries out obtaining capacitance signal.If detected target substance is bacterium, the relative dielectric constant of these bacterium target substances is the magnitudes 100, and ferrofluid by 80 magnitude, so, their existence in ferrofluid can detect, if cell can be positioned at the position close to sensing electrode.
Therefore, by using the magnetic field produced, such as, use and configure about those the similar electrodes described in Fig. 1-16, target substance can push channel roof to, and sensing electrode can be positioned herein.In some embodiments, the detecting device used in conjunction with microfluidic devices and system comprises antibody " blanket " (specifically target acquisition cell), and a pair capacitance detector---catching blanket upstream for one, another is in downstream.In such an arrangement, sensing capacitor can count and enter (
n_in) and leave (
n_out) capture region cell number and according to calculating poor (that is,
n_in-N_out) define and how much caught by blanket.Alternatively, the electrode pair of measurement impedance can be used, or measurement represents some other devices of some other character of the number/amount of individual target material.Implement micro-fluidic system as herein described and equipment is enough strong with the shear flow obtained on the surface at antibody blanket, to prevent the non-specific attachment of other cell, but be not too strong to such an extent as to the loss of target cell can not be caused.In this operator scheme, different capture regions can be placed on downstream each other continuously, and be multiple target substance, such as, pathogen or other target cell such as CD4+ white blood corpuscle, the concrete cancer cell etc. of expressing epithelial cell adhesion molecule (EpCAM) provides quantitative with concrete detectability.
Antibody blanket can be realized by various functionalisation of surfaces.Sensing electrode can be printing, pattern transfer printing or micro-machined (evaporation of the metal on photoetching pattern surface or sputtering).
Some advantages of sensor-based microchannel comprise:
To be separated with actual cell sensor (detecting device) by making functionalisation of surfaces and to simplify manufacture process.
The biology sensor direct function sensor surface of the much common existing electron device with integrating.According to micron or nanoscale sensor (it is said and provide much higher sensitivity), functionalized total surface area is very little.Sometimes, a few cell is only had also to have living space to sensor surface is no more than.Therefore, if target cell concentration is higher, the saturated fast of sensor signal can be ensured.In order to address this problem, its sensor is arranged to wide array (nearly thousands of) by some common devices, causes the complicacy of system and the challenge of manufacturability significantly to increase.In the system based on miniflow described herein and device, use the detecting device of these systems and device can detect lacking to individual cells through surveyed area; The maximum number of the cell that can be detected before signal is saturated is provided by the relative extent of catching blanket.The dynamic range of the sensor used in the micro-fluidic system disclosed in increase and device simply to the scope increasing capture region, and can not need the number increasing sensor.
Detection method as herein described is unmarked (that is, unstressed configuration mark or other mark are attached on cell and allow to detect).
Use (or the use of some other magnetic field generation mechanism of current-carrying electrodes concentrating stream of cells and push to surveyed area below passage, such as, mechanism according to permanent magnet), input pressure sampling stream can be used, in certain embodiments, be less than 1 psi(pound per square inch), keep good flux simultaneously.On the contrary, in common cell manipulation, the height suitable with cell dia (typically 50 microns or less) and the passage of width must be used, and thus or the flux of those devices lower, or very large source pressure must be used to promote to flow through these small passages fast.
Therefore, with reference to Figure 17, the schematic diagram of the part of an example embodiment of microfluidic devices 900 is shown.Device 900 comprises microchannel 910, and it can be similar to, such as, about Figure 1A, and the microchannel described in 4,5,13 and/or 14.Device 900 comprises one or more detecting device 920a-n further, the side of position adjacent channel 910 is (in the embodiment described, detecting device is positioned at the outer wall of passage 910, the top of its define channel), be configured to detect and determine to flow through existence and the number of target substance in microchannel 910 sample, and determining type or the identity of detected target substance.Device 900 also comprises magnetic field generation mechanism and pushes the target substance in ferrofluid sample to microchannel 910 top to produce, and thus pushes the magnetic field of detecting device 920a-n to.In some embodiments, magnetic field generation mechanism can use one group of electrode to implement, such as, shown in electrode 930(dotted line), it can be similar such as about shown in Fig. 1-17 and described those.These electrodes can have predetermined configuration, make when at least one controlled electric current produces controlled magnetic field by during these electrodes, nonmagnetic target substance (cell, bacterium etc.) is pushed to the microchannel surface relative with near the surface of electrode by it.
Such as, electrode can comprise at least two electrodes of the contiguous microchannel in position, it is configured to conduct at least one electric current that controllably provides and produces controlled magnetic force, this at least in part according to the physical attribute of electrode, the physical attribute of microchannel and/or apply the attribute of electric current.These physical attributes can comprise the structure of at least two electrodes, the structure of wherein said at least two electrodes can comprise following one or more, such as, the location of the one or more substantially straight shape of at least two electrodes, the substantially corrugated shape of the one or more of at least two electrodes, the substantially parallel layout of at least two electrodes and/or at least two electrodes taper substantially, wherein said at least two electrodes are close to each other gradually.Electrode is configured to work as the controlled electric current of at least one, and (it can use controller, the controller 160 of such as Figure 1A controls) when being applied at least two electrodes, controlled magnetic force is produced in the sample comprising ferrofluid, described electric current comprises association attributes (such as, phase place, frequency, amplitude etc.).
In some embodiments, permanent magnet can be used to supplement or alternative electrode, to promote that producing magnetic field pushes to target substance from the nearest channel surface of detecting device 920a-n.
As described in, and as shown in figure 17, the detecting device based on electric capacity, the detecting device etc. based on impedance can be used in some embodiments.Such as, detecting device 920a comprises upstream capacitance detector 922a, comprise two sensing electrode plates, capture region (also referred to as blanket district) 924a, with be positioned at the downstream capacitance detector 926a in capacitor 922a downstream and blanket 924(downstream capacitance detector 926a also as upstream capacitance detector, be labeled as 922n), for the detecting device that next continuous print uses in conjunction with the microfluidic devices 900 of Figure 17.
Upstream capacitance detector 922a be configured to the number (or approximate number) of the target substance (as cell, bacterium etc.) determining to exist in sample the electric capacity that produces.Based on the electric capacity detected (or impedance), qualification unit 940(its can be calculation element based on processor), may be used for the number (or approximate number) determining to enter detecting device
n_in.With reference to Figure 18, show the schematic diagram of capacitance detector and the description bacterium figure of capacitance variations by causing near detecting device face.As illustrated in the figure of Figure 18, the number through bacterium (or some other target substances) is larger, and detected electric capacity is larger.
By upstream capacitance detector 922a determine through target substance number after, target substance flows through near antibody blanket 924a, and it functionalised to catch, and thus detects the target substance (cell or some pathogen) of particular type.Blanket 924 helps to obtain specificity in the detection of target substance, and target substance can have similar size and structure (this makes visual assessment, such as, use microscope, more difficult and uncertain).Such as, Escherichia coli and Salmonella are shaft-like, have similar Size Distribution, but these target substances can use the antibody for functionalized one or more antibody blanket to be distinguished.
As described in, target substance can be pushed to blanket 924a enough close, to realize more effective capture ability by the use of magnetic field generation mechanism (electrode or permanent magnet).Subsequently, the target substance of not catching continues flowing and through downstream capacitance detector 926a, and it measures the electric capacity (or alternatively, impedance) remaining the target substance of not catching and cause, then based on the determined electric capacity of downstream capacitance detector 926a, can determine remain target substance number (
n_out) (such as, being tested and appraised unit 940).
n_inwith
n_outdifference thus the number of the target substance of being caught by blanket 924a can be obtained.In some embodiments, the determination of the individual member of institute's capturing target substance shows to there is target substance in sample, if
n_inwith
n_outbetween difference exceed predetermined threshold value.Also the testing mechanism (such as, the testing mechanism based on impedance) of other type be can use, quantity and the identity of the various target substances in the sample flowed determined similarly.
In some embodiments, the series connection target cell detecting device that similar (or even not same) can be used to configure.Such as, in the embodiment shown in Figure 17, meet continuous print detecting device 920b after the first detecting device 920a, its blanket district (that is, capture region) can be configured, and (by suitable functionalized) catches, and thus detects dissimilar target substance.
Therefore, in some embodiments, the detecting device that microfluidic devices as herein described and system use can comprise the electrode at a pair interval separately, to measure the electric capacity (or impedance) in microchannel, with qualification unit, with the existence based on measured electric capacity determination at least one target substance.The existence of at least one target substance is determined in the change of the electric capacity in the microchannel measured by qualification unit can be configured to cause based on the existence due at least one target substance.In some embodiments, detecting device can be configured to measure other character of affecting by the existence of target cell, such as impedance etc.Each detecting device may further include capture region, it comprises and is configured to material interactional with one of plurality of target material, capture region is positioned at the microchannel downstream of the electrode pair at interval, and another downstream electrode at interval being arranged in capture region is to measure the electric capacity (or impedance) of microchannel.
As described in, in some embodiments, identify that cell location is determine the initial number of at least one target substance in the electrode pair at interval, and determine that at least one target substance is at another end number to the electrode place at interval, and determine whether the level of at least one target substance of catching in captured region exceeds predetermined threshold value based on the difference between initial and end number at least partly.
As further described, in some embodiments, the test set that the order that detecting device comprises series connection is arranged, the test set that the order of each series connection is arranged comprises the electrode at first pair of interval to measure the electric capacity (or impedance) in microchannel, capture region comprises and is configured to material interactional with one of plurality of target material, and capture region is positioned at the downstream of the electrode at first pair of interval.The test set that the order of each series connection is arranged also comprises the electrode at the second pair of interval being positioned at capture region downstream to measure the electric capacity (or impedance) of microchannel, with qualification unit is to determine the initial number of at least one target substance at the electrode place at first pair of interval at each test set place, with at least one target substance is at the end number at the electrode place at second pair of interval, and determine whether the level of at least one target substance of catching in captured region exceeds predetermined threshold value based on the difference between initial and end number at least partly.
With reference to Figure 19, show the figure of the example procedure 1000 detecting at least one target substance in sample.Method comprises: 1010 accept to comprise at microchannel (such as shown in Figure 1A, 4,5,13,14 and 17) and are suspended at least one target substance in biocompatible ferrofluid (such as, cell, bacterium, non-organic body material etc.) sample, such as described herein those.
After accepting sample, 1020 at least one electric currents are controllably applied (such as, use controller, such as based on the controller of processor, Non-follow control etc.) at least two electrodes (or being even unitary electrode) of contiguous microchannel to position, to produce controlled magnetic force in the ferrofluid passage containing sample, make described at least one target substance guiding detecting device.Electrode can be any electrode configuration relating to such as Fig. 1-18 described herein.In some embodiments, detecting device can be based on capacitance measurement, or based on measuring the detecting device comprising other character (such as, impedance) of the sample of target substance.This detecting device can determine the number (or measuring) of target substance in the input and output stage of capture region (such as, with being configured to and the blanket of the specific antibodies functionalization of specific objective matter interaction that exists in sample).The number being configured the target substance member (such as, single organism) that (such as, functionalized) catches for the capture region of catching specific objective material type can be shown in the difference of the target substance number of input and output phase measuring.Therefore, based on the measurement that detecting device carries out sample in microchannel, at least one target substance in sample is determined 1030.
With reference to Figure 20, the schematic diagram of display general-purpose computing system 1100, it can be used for implementing any system/device based on processor, can be used in conjunction with system as herein described and equipment, comprises the qualification unit 940 etc. of the controller 160, Figure 17 of Figure 1A.This computing system 1100 comprises the device 1110 based on processor, such as personal computer, special computing equipment, etc., it typically comprises central processor unit 1112.Except CPU1112, system comprises primary memory, cache memory, bus interface circuit (not shown).Device 1110 based on processor comprises massive store element 1114, the hard disk drive be such as associated with computer system.Computing system 1100 may further include keyboard, or keypad 1116 and display 1120, such as, and CRT(cathode-ray tube (CRT)) or LCD(liquid crystal display) display.
Device 1110 based on processor is configured to be conducive to, such as, control as herein described and process operation, such as, determine and/or control to be applied to the controlled electric current of at least one of electrode, to produce controlled magnetic field, based on detected character (such as, electric capacity, impedance) determine the number/amount of target substance, etc.Memory device 1114 thus also can comprise computer program, when causing the device based on processor to operate based on when the device 1110 of processor performs, is beneficial to implement control as herein described and/or process operation.Device based on processor can also comprise the peripheral unit exercising input/output function.These peripheral units can comprise, such as, CD-ROM drive and/or flash drive, or network connects, for downloading related content to connected system.These peripheral units can also be used for the software of download package containing computer instruction, make respective system/device carry out general operation.Alternatively and/or additionally, in some embodiments, the logical circuit of special object, such as, FPGA(field programmable gate array) or ASIC(application specific integrated circuit) may be used for the enforcement of system 1100.With the described device 1110 based on processor can by together with other module of comprising be loudspeaker, sound card, pointing device, such as mouse or trace ball, user utilizes them to provide and is input to computing system 1100.Device 1110 based on processor can comprise operating system, such as: the operating system of Windows XP Microsoft.Alternatively, other operating system can be used.
In addition, various illustrative components, blocks, module and the method related to described in embodiment disclosed herein can use general processor, digital signal processor (" DSP ") or other programmable logic device, discrete gate or transistor logic, discrete hardware components or its any combination being designed to perform function described herein implement or perform.General processor can be microprocessor, but in replacement scheme, processor can be any processor, controller, microcontroller or state machine.Processor can also be implemented as the combination of calculation element, such as, the combination of the combination of DSP and microprocessor, multi-microprocessor, one or more microprocessor and DSP kernel or any other this type of configure.
Computer program (also referred to as program, software, software application or code) comprises the machine instruction for programmable processor, and with high-level program and/or OO programming language, and/or can implement with assembling/machine language.As used herein, term " machine readable media " refers to any non-transitory computer program product, equipment and/or device (such as, disk, CD, storer, programmable logic device (PLD) (PLD)), for providing machine instruction and/or data to programmable processor, comprise the machine readable media of non-transience, it is accepted as the machine instruction of machine-readable signal.
Mutual in order to what provide with user, theme described herein can be implemented on computers, described computing machine has display device (such as, CRT(cathode-ray tube (CRT)) or LCD(liquid crystal display) display) for showing information to user, and keyboard and pointing device are (such as, mouse or trace ball), utilize its user to provide and be input to computing machine.It is mutual that the device of other kinds also can be used to provide with user, and such as, the feedback being supplied to user can be any type of sensory feedback (such as, visual feedback, audio feedback or tactile feedback); And the input of user can be received in any form, comprise sound, voice or sense of touch input.
Partly or entirely can the comprising aft-end assembly (such as data server) or comprise middleware component (such as of theme as herein described, apps server) or comprise front end assemblies and (such as there is graphic user interface or Web browser, client computer by its user can be mutual with the embodiment of theme described herein), or implement in the computing system of any combination of these rear ends, middleware or front end assemblies.The assembly of system can be interconnected by the digital data communication (such as, communication network) of any form or medium.The example of communication network comprises LAN (Local Area Network) (" LAN "), wide area network (" WAN ") and internet.
This computing system can comprise client and server.Client and server is general all away from the other side, and interactive typically via communication network.Relation between client and server usually by means of operating on respective computing machine, and has the computer program of client-server relation each other and produces.
Various illustrative components, blocks, module, circuit and relate to above-mentioned figure and the method described in embodiment disclosed herein, often may be embodied as electronic hardware, computer software or both combinations.
In addition, relate to the method/program described in embodiment disclosed herein, can hardware be embodied directly in, the software module performed by processor, or in both combinations.Software module can reside in the storage medium of RAM storer, flash memory, ROM storer, eprom memory, eeprom memory, register, hard disk, removable dish, CD-ROM or other form any, comprises in network storage medium.Exemplary storage medium can be coupled to processor, and this processor from its reading information, and can be write information in storage medium.In replacement scheme, this storage medium can be integrated into processor.Processor and storage medium also can reside in ASIC.
Describe some embodiments of the present invention.But it will be understood to, various modification can be carried out and without departing from the spirit and scope of the present invention.Therefore, other embodiment is in the scope that following patent requires.
The calculating of annex A---particle manipulation
Provided herein is that the particle speed that makes to observe in ferromagnetic microfluid system described herein, apparatus and method and threshold frequency can by the analytical approachs of evaluation.In order to simplify calculating, assume ideally spherical incompressible particulate, Magnetic linear ferrofluid, and independent of the ratio of slip (seeing below) of magnetic field intensity and frequency.The radius of further supposition spheric grain
(R)compare determined by electrode size and spacing travelling-magnetic-field (
h → ) wavelength be little, make
r|
h → | << |
h → | set up.According to these hypothesis, the ferrofluid magnetization of next-door neighbour's particulate---with the volume of particle
vpthe interior effective magnetization
m eff ---it is uniform for can being similar to.Also approximate, any value (but not being its gradient) is constant at the Nei Yannei of particulate.
Then, the total transient force on this dipole is provided by following equations
(equation A)
Wherein
b in the magnetic flux density in spherical particle, and to granule interior volume integral (Zahn etc., 1995,
j of Magnetism and Magnetic Materials149:165-173).According to these hypothesis, the surperficial item of equation A, due to
mwith
buncontinuity to produce integration be the pressure term of 0, the simple vector of integrand expands display, and this is identical with Kelvin (Kelvin) force density.Therefore, transient force expression formula can be reduced to
(equation B).
In order to obtain the analysis expression of final magnetic force, in the non-existent situation of particulate according to external magnetic field (
h ext ) represent
m eff and
b in helpful, because this value easily can obtain from simple simulation.(have complicated, the magnetic permeability of frequency dependence in ferrofluid
μ p= μ 0 (1+χ
f )) there is magnetic permeability
μ p (be substantially
μ 0 ) the net magnetization of particle depend on as follows
h ext
(equation C).
Determine that intragranular magnetic flux density and magnetic field need to consider the demagnetizing field of its inside.Intragranular resultant field for spheroid is
h → in =
h → ext -
h → dmag , with
h → dmag =
m → eff / 3.Therefore, under linear condition, granulated magnetic can be written as
m → eff =χ
eff h → in , can obtain
(equation D).
Comparison equation C and D shows the effective granulated magnetic rate in ferrofluid magnetic susceptibility:
(equation E).
It should be pointed out that effective magnetic susceptibility depends on ferrofluid, because particulate response magnetic force is only because it replaces ferrofluid, and produce " bore ".In this respect, the magnetic medium at place, hole determines the interactional intensity between bore and the field applied.Negative sign in equation E shows that the effective magnetization of particulate is the direction that local ferromagnetic fluid magnetic is contrary in a static condition.Although for χ
eff ≈ χ
f , for χ
f << 1, the effective susceptibility of bore tends to-1 in hard magnetization medium.In fact, overgenerous ferrofluid is used to run counter to desire for particle manipulating.
Instantaneous magnetic force on particle can be expressed as
(equation F).
Here,
θbe
m → eff with
h → in between angle, by resultant magnetization rate χ
eff angle provide.Use
, we obtain
(equation G).
When there is row field, local magnetic field sinusoidal variations, and time average power is only the half of transient force maximal value:
(equation H).
Similarly, the instantaneous torque on magnetic dipole is provided by following equations
(equation I)
(Zahn?et?al.,?1995,?
J?of?Magnetism?and?Magnetic?Materials?149:165-173)。At this, the rotation of non-magnetic particles may be subject to the ratio of slip between 0 and 1
sthe possibility of impact be allowed to.The ratio of the value of torque that the isolated particle of the torque that the non-magnetic particles that represents this ratio of slip experiences in ferromagnetic microfluid system described herein and device and same size and effective magnetizing will be experienced.
To all the other detailed derivations of magnetic moment and magnetic picture with.Replace magnetic flux density, obtain
(equation J).
Due to
, obtain
(equation K).
Time average torque is provided by following formula
(equation L).
For given input current amplitude, program of finite element (COMSOL) is used to calculate
h ext , use the realistic two-dimensional section of ferromagnetic microfluidic channel and lower electrodes.The Reynolds number (Reynolds'number) relevant with the motion of cell in static ferromagnetic fluid to micro-meter scale pearl is very little.In this scheme, the impact of inertia is negligible, stokesian fluid equation major flow body dynamics.Therefore, the balance between viscous drag and magnetic force determines granular dynamics.Because stokesian fluid equation is linear, all fluid dynamics coefficients related to can be incorporated in resistor matrix:
(equation M)
(Happel?J,?Brenner?1-1?(1983)?Low?Reynolds?Number?Hydrodynamics?with?special?applications?to?particulate?media.?(Martinus?Nijhoff:?Dordrecht))。Herein, ν is the linear velocity of particulate along passage length, and ω is its angular velocity, and η is ferrofluid viscosity,
rmicrosphere radius, and
f i be depend on particle radius and itself and channel roof spacing (
h) the resistance factor.Suppose
h << < R,these resistance factors can from standard lubrication theory obtain into
(equation N)
(Goldman?et?al.,?1967,?
Chem?Eng?Sci?22:637-651)。Usually, likely by Dedaguin, Landau, Verwey and Overbeek theory (dlvo theory) (Ise, 2007,
proc Jpn Acad B Phys. Mal Sci83; 192-198), use on particulate and estimate with the surface charge density of channel surface
it, consider the ionic conditions in ferrofluid simultaneously.Ironically, particulate is pushed to the vertical direction of channel roof power (
f avg, y ) be the magnitude of nN, and expect that they will close to contact channels wall.
Equation M can ask ν and ω by a simple matrix inversion,
(equation O)
Wherein
(equation P).
Here,
g=f l f 4 -f 2 f 3 be defined according to the facility on symbol.Therefore, because the particle linear velocity of magnetic force and torque can be determined separately:
(equation Q and R)
Clean particle speed is then provided by following formula
(equation S).
Magnetic force and magnetic moment with particle volume (
r 3 ) weigh; According to equation Q and R, be clear that the particle speed due to magnetic force depends on
r 2 , and weigh due to the particle speed R of magnetic moment.This observations shows, the impact of magnetic moment on less particle is relatively more remarkable, and explains why less particulate shows less threshold frequency at the dynamically middle of them.
Aforesaid theoretical method explains the experimental result (such as, Figure 10 A) of the h of ratio of slip 1 and about 1nm well, confirms that particulate is pushed to the expection of channel roof really consumingly.Ratio of slip 1 represents that microballoon rotates under nonslipping condition.
Accessories B---the power on magnetic dipole
Usually, the magnetic force on magnetic dipole can use Kelvin Force expression formula to find, namely
(equation T).
This expression formula is about as much as the equation A shown in appendix A.Crucial hypothesis is, the field applied is not too uneven and particle radius (
r) enough little, make
r|
h → | << |
h → | in any direction.According to this hypothesis, the magnetization of the ferrofluid immediately around particulate can be thought uniformly.Approximate further, any value (but not being its gradient) is constant at the Nei Yannei of particulate.
Remember that the integral function that these hypothesis simplified, vectorial may be used for rewriting equation A is as follows:
(equation U).
The Section 1 of equation U right-hand side (RHS) relates to the curling of magnetic flux density, can expand as
(equation V).
In the integration volume of equation A, the curling of magnetic field of any position is 0, because ferrofluid and plastic particles are insulation and do not support electric current.Therefore, the Section 1 of the RHS of equation V disappears.Interparticle magnetized curling be 0, but on its surface, effective magnetization is as a step change.Therefore, should usually be considered to the surface contribution of force density.But because the magnetization of the ferrofluid around immediately particulate is assumed that it is constant, when around spheroid during integration, the Section 2 of equation V also disappears (due to symmetry).Same logic can be applied to the B in equation U
→ x (
x M
→ ) item: microballoon inside (
x M
→ ) be 0, and around ball surface B
→ x (
x M
→ ) integration is 0, the magnetic flux density at next-door neighbour microballoon place and ferrofluid magnetization supposition constant.
Based on same reason, (the B in equation U
→ .
) M
→ item will also be 0 in microballoon inside, and integration is 0 around it.The item then uniquely relating to the non-zero gradient of field vector is
(equation W).
Effective to the volume integral of non-magnetic particles.Therefore, at interparticle
b → =
μ 0 h → , and equation W becomes same with equation T-phase.In other words, according to the hypothesis emphasized above, equation A and T is of equal value when setting shown in this article.
Equation A can be used as the expression formula of power, instead of in equation T, because the former causes power, its direction is determined by gradient operator, needs to take single derivative along given direction in space to determine the power along this direction.
It is also contemplated that what relevant surface graded of expression formula there occurs in equation A.Again, use the magnetization of ferrofluid and magnetic flux density around microballoon is inner and surperficial (but not being their derivant) to be this constant hypothesis, it can be defined as
(equation X).
Here, field and magnetization vector on x-z plane, due to the symmetry of ferromagnetic microchannel.As previously mentioned, the expression in equation X is only evaluated at interparticle, to calculate the power in x direction.If do not have general any loss, the center of microballoon is taken as initial point.
m → with
b → be discontinuous on particle-ferrofluid border, therefore their derivant causes pulse, when when microsphere surface integration,
m x, out b x, out – M z, in b z, in to from x=√ (
r 2–
y 2–
z 2) place surface patch integration each contribution by x=-√ (
r 2–
y 2–
z 2) negative contribution of the relative small pieces in place offset.Result surface integration is 0.Due to spheroid symmetry, right
f z in item be also like this.Therefore, according to described hypothesis, at interparticle equation A evaluation obtained to the magnetic force on it.
Claims (18)
1. the device of the plurality of target material of separate out suspended in biocompatible ferrofluid, described device comprises:
Biocompatible ferrofluid;
Microchannel, comprise at least one sample inlet and at least one outlet, this microchannel has the passage length extended between at least one sample inlet and at least one outlet, this microchannel is configured to accept continuous print sample stream substantially from least one sample inlet, this passage is configured to make sample flow at least one outlet along passage length, described sample comprises plurality of target material, and described target substance is suspended in biocompatible ferrofluid; With
Position is close to microchannel and the multiple electrodes be substantially arranged in parallel with microchannel, and multiple electrode is configured to produce magnetic field model when electric current is applied to multiple electrode along the passage length at least partially of microchannel,
Wherein magnetic field model be configured to when sample stream along cause during the advancing at least partially of microchannel at least two kinds of plurality of target material in sample stream separated.
2. the device of claim 1, it comprises power supply further, and described power configuration is for controllably to apply electric current controllably to produce magnetic field model to multiple electrode.
3. the device of claim 2, one or more electric current of wherein said power configuration selected amplitude, institute's selected frequency and/or selected phase place for applying has, wherein sample at least two kinds of target substances be separated to small part based on one or more of amplitude, institute's selected frequency and selected phase place selected by electric current.
4. the device of claim 1, wherein said multiple electrode is configured to produce controlled magnetic force component and controlled magnetic moment component.
5. the device of claim 1, wherein said multiple electrode is configured to cause at least two kinds of target substances being separated with on the direction of the direction perpendicular of continuous print sample stream substantially in microchannel.
6. the device of claim 1, it comprises stream generation unit further, this stream generation unit is configured to produce continuous print stream substantially, described stream generation unit comprises following at least one: forcing pump, syringe pump, peristaltic pump, vacuum plant, the structure flowed by gravity, and produces the device of capillary force.
7. the device of claim 1, wherein said sample comprises the material based on cell.
8. the device of claim 1, wherein said biocompatible ferrofluid comprises appropriate ionic species further to control osmotic pressure on cell to promote the continuation of cell.
9. the device of claim 8, wherein said biocompatible ferrofluid comprises the citric acid salt concentration of about 5-200mM further.
10. the device of claim 9, wherein said biocompatible ferrofluid comprises the citric acid salt concentration of about 40mM further.
The device of 11. claims 9, wherein said biocompatible ferrofluid comprises the ionic strength of the design of about 150mM further, is the eukaryotic that isotonic and applicable maintenance is lived to make biocompatible ferrofluid.
The device of 12. claims 1, wherein said biocompatible ferrofluid has the pH of about 7.4.
The device of 13. claims 1, wherein said at least two kinds of target substance based target sizes are separated.
The device of 14. claims 1, wherein said at least two kinds of target substance based target shapes are separated.
The device of 15. claims 1, wherein said at least two kinds of target substance based target elasticity are separated.
The device of 16. claims 1, wherein said at least two kinds of target substance based target forms are separated.
The device of 17. claims 1, wherein said at least two kinds of target substances based on electrode separation, apply the frequency of electric current, and apply the phase place of electric current one or more be captured.
The method of 18. separation plurality of target materials, described method comprises:
The sample stream that continuous print substantially comprises the multiple target substance be suspended in biocompatible ferrofluid is accepted at the entrance of microchannel;
Sample is passed through along microchannel; With
To position adjacent channel and substantially parallel with passage multiple electrodes apply the controlled electric current of at least one, described current arrangements is controllably produce magnetic field model along the passage length at least partially of microchannel, to cause when sample stream is separated along at least two kinds of plurality of target material in sample during the advancing at least partially of passage length.
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| AU (1) | AU2011320908A1 (en) |
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| US20130313113A1 (en) | 2013-11-28 |
| CN104535783B (en) | 2017-05-24 |
| EP2633330A1 (en) | 2013-09-04 |
| US9999855B2 (en) | 2018-06-19 |
| EP2633330A4 (en) | 2018-01-10 |
| CN104535783A (en) | 2015-04-22 |
| JP2014500949A (en) | 2014-01-16 |
| EP2633330B1 (en) | 2022-05-11 |
| WO2012057878A1 (en) | 2012-05-03 |
| JP5809282B2 (en) | 2015-11-10 |
| AU2011320908A1 (en) | 2013-05-23 |
| EP4105656A1 (en) | 2022-12-21 |
| CN103403557A (en) | 2013-11-20 |
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